At the courthouse in Hillsboro, a small Southern town, Bertram Cates is behind bars, awaiting trial for teaching his students about Darwin’s theory of evolution. Rachel Brown, a friend and fellow teacher who also is the daughter of the town’s minister, visits Cates. She brings Cates some clean clothes and urges him to plead guilty and throw himself at the mercy of the court. Cates remains firm in his resolve.

Hillsboro erupts with excitement as prominent lawyers and journalists arrive for the trial. E. K. Hornbeck, a critic for the Baltimore Herald, surveys the scene and makes wisecracks. The Bible-thumping politician Matthew Harrison Brady, who leads the prosecution, arrives to a warm welcome from the townspeople and a picnic in his honor. Brady meets with Reverend Brown, District Attorney Tom Davenport, and the mayor. Brady also holds a confidential discussion with Rachel about her friendship with Cates. Rachel leaves the discussion feeling that she has betrayed her friend.

Hornbeck informs the crowd that the prominent litigator Henry Drummond will represent the defense. The mayor names Brady an honorary colonel in the state militia. Reverend Brown and the mayor discuss how they might prevent Drummond from entering Hillsboro. When the crowd disperses, Rachel and Hornbeck discuss Hornbeck’s columns, which portray Cates as a hero. Around sunset, Hornbeck greets Drummond, who has just arrived in town.

A few days later, Drummond, Brady, Davenport, and the judge conduct jury selection. They accept the illiterate Mr. Bannister. Brady makes a joke about Drummond’s bright purple suspenders, but Drummond turns the tables by revealing that he bought the suspenders in Brady’s Nebraska hometown. As jury selection continues, Brady rejects Mr. Dunlap, a fervent supporter of Brady. Drummond mockingly objects to Brady’s honorary title of “colonel,” so the judge grants Drummond the same title to even the score. Brady and Drummond accept Sillers, a feed store employee, as a juror. Drummond, who argues that the evolutionist movement should be given the same amount of attention as the fundamentalist movement, notes that the townspeople have erected a sign commanding “Read Your Bible!” in the town square and have advertised prayer meetings. Frustrated by Drummond’s demands, the judge declares the court in recess.

A crowd of admirers surrounds Brady as he leaves the courtroom, but no one dares to come near Drummond. Before Drummond leaves the courtroom, Rachel expresses to him her concerns about the trial. Cates relates the hardships he has endured since his arrest. Drummond, who empathizes with Cates’s struggle and isolation, offers Cates the opportunity to change his plea on one condition: that Cates truly believes he has done wrong. Cates decides to persevere for his cause. Rachel, however, informs them that Brady has asked her to testify against Cates. A frantic Cates returns to his cell, concerned about the details of personal conversations that Rachel might betray. Drummond reassures Rachel that Brady is less powerful than she believes. Assuring her that Cates is fighting for a worthy cause, Drummond compliments Rachel on her strength in loving Cates.

On the courthouse lawn, Brady leads a group of reporters to a prayer meeting that Reverend Brown is about to conduct. Brady tells the reporters about his former friendship with Drummond. Brown begins the prayer meeting with a quick recitation of the creation story presented in Genesis. He proceeds to incite the crowd into a frenzy. The climax of Reverend Brown’s rant is an incantation to bring the fires of hell down on Cates. When Rachel protests, her father requests the same curse for her. Brady, disturbed by Brown’s zeal, interrupts and reminds the preacher of the Christian doctrine of forgiveness. Brady calls the prayer meeting to an end and then speaks to Drummond about their old ties and how they have drifted apart.

Two days later, in the courtroom, Brady questions thirteen-year-old Howard Blair about Cates’s teaching. Howard confirms that Cates taught him that humans descended from “Old World Monkeys” and that his teachings on creation omitted any reference to God. Cross-examining Howard, Drummond asks the boy about evolutionary theory. The prosecution objects, but Drummond claims he is trying to establish the basic human right to think. The prosecution and the judge counter that the trial is not about the right to think. Drummond asks Howard whether evolutionary theory has harmed him, and the question confuses the boy. Drummond asks Howard if he believes what Cates taught him. Howard says he hasn’t made up his mind. Drummond then asks the boy what he thinks of modern technological advances that are not mentioned in the Bible. Howard is again confused, and Brady objects. Drummond rails against Brady’s absolute notions of right and wrong. Drummond asks Howard if he understands what is being discussed. When the boy says no, he is dismissed.

The prosecution calls Rachel to the stand. To explain why Cates stopped attending church, Rachel tells the story of Tommy Stebbins. An intellectually curious boy, Stebbins was one of Cates’s favorite students. When Stebbins drowned in a local river, Rachel’s father preached that the boy would suffer eternal damnation because his parents never had him baptized. Upset both by the death of the boy and the preacher’s reaction, Cates stopped going to church. Brady asks Rachel further questions about her discussion of Cates’s ideas, but Rachel falters and becomes visibly upset. The prosecution rests its case.

Drummond attempts to call several expert scientists to testify about evolutionary theory, but the judge says that their testimony is inadmissible. Drummond shifts gears and calls Brady to the stand as an expert on the Bible. Asking Brady several questions about Biblical passages that defy the tenets of modern science, Drummond catches Brady off balance and gains the support of the crowd. As Drummond exposes contradiction after contradiction in Brady’s views, Brady becomes hysterical and begins to shout names from the Bible. Davenport objects, and the judge adjourns the court.

The next day, just before the jury reads its verdict, Cates and Drummond discuss Cates’s chances. A radio reporter enters the courtroom to set up his equipment. The mayor takes the judge aside and tells him that political forces in the state are growing worried about media coverage of the trial. The mayor implicitly tells the judge to pass a light sentence. The jury hands their verdict to the judge, who declares Cates guilty and fines him $100. The prosecution objects to the light sentence. Drummond demands an appeal, and the judge grants him thirty days to prepare it.

The judge adjourns the court. Brady tries to read some prepared remarks, but the spectators in the courtroom begin to leave. Brady tries to deliver the speech to the radio reporter, but the reporter says that the station producer has cut him off. Brady has a mental breakdown and must be carried out of the courtroom as he deliriously recites what sounds like a presidential campaign victory speech. Hornbeck mocks Brady, while Cates expresses concern for him.

Meeker, the bailiff, tells Cates that Hornbeck and the Baltimore Herald have posted $500 for Cates’s bail. Rachel tells Cates that she has decided to leave her father and that she has overcome her fear of thinking for herself. Word arrives that Brady has died of a “busted belly,” and Cates, Rachel, and Drummond decide to leave together on the train out of town that evening.