Uncle Vanya I opens on a muggy autumn afternoon in the garden of Professor Serebryakov's estate. Marina, an old nanny, sits by a samovar as Astrov, the country doctor, reminisces about the time when he first came to the region, a time when Vera Petrovna—Serebryakov's first wife and mother to his daughter Sonya—was still alive. Serebryakov has recently returned with his beautiful young wife, Yelena, to live on the estate; Astrov has come to treat the Professor's case of gout.

Astrov delivers an extended speech about how life has become "boring, stupid, sordid" and how his feelings are "dead to the world"; he needs nothing, wants nothing, and loves no one. Voynitsky (or Uncle Vanya), Serebryakov's brother-in-law by his first marriage and caretaker of the estate, then enters, yawning. He complains that the professor and his wife have thrown the estate out of kilter: everyone has succumbed to lethargy.

When Astrov reproaches Voynitsky for his ill humor, Voynitsky replies that Voynitsky has grown old and lazy, having wasted his entire life. For many years Voynitsky—who once worshipped the professor—has sent the farm's proceeds to the professor, while reserving only a beggar's salary for himself. He now considers the professor a charlatan. When Astrov remarks that Voynitsky seems to envy Serebryakov, he readily concurs, in particular when it comes to his success with women.

Sonya, Yelena, and Maria Vasilevna (Voynitsky's mother) join the party. Astrov invites Yelena and Sonya to his forest preserve. Though admitting that perhaps only an "eccentric" could think thus, he then decries man's impulse to destroy, extolling the beauty of nature and man's capacity leave his legacy to future generations. Yelena and Voynitsky then walk to the veranda. Voynitsky protests Yelena's marriage and suddenly attempts to declare his love to her: she rejects him wholeheartedly.

Act II takes place at night with the professor and Yelena sitting next to each other in the dining room, asleep. A night watchman can be heard tapping in the garden. The two awaken, and Serebryakov complains of his old age. He has spent his life in scholarship only to end up in "exile." Voynitsky arrives to relieve Yelena: the professor reacts in terror. Marina enters and tenderly takes Serebryakov to bed, leaving Yelena and Voynitsky alone.

Much to Yelena's dismay, Voynitsky resumes his attempts at seduction. When Yelena recoils, he once again decries the years he has wasted; Yelena is numb to his entreaty. She leaves, and Voynitsky delivers a soliloquy that imagines what could have been had they married when they first met. Some disconcerting comic relief ensues involving a tipsy Astrov and guitar-playing Telegin (an impoverished landowner dubbed "Waffles" for his pockmarked face). Once Voynitsky has exited, Sonya, who has entered the room in the meantime, converses with the brooding Astrov. The doctor moans that he cannot love but is "fascinated" by beauty (namely Yelena's). When Sonya hypothetically asks him what he would do if she knew someone who loved him, he answers that could not love her in return.

Once alone, Sonya confesses her love for Astrov. Yelena then enters. The two women suddenly exclaim that they must reconcile—apparently they had been at odds since Yelena married Sonya's father even though she does not love him. Inexplicably, Sonya begins laughing, exclaiming that she is happy. Yelena impulsively decides to play the piano, and Sonya rushes out to ask her father's permission. Unfortunately it is withheld.

Act III opens in the house drawing room with Voynitsky and Sonya seated as Yelena paces about. The professor has called a meeting. Sonya criticizes Yelena's "infectious" idleness for causing everyone to desert his or her work. Yelena is enraged: Voynitsky offers to pick her a bouquet of roses.

Once Voynitsky exits, Sonya expresses her anguish over the doctor anew, and Yelena resolves to find out if he loves her. After Sonya runs to fetch him, Yelena, now alone, confesses her own fascination for Astrov. Astrov then enters with a cartogram and proceeds to explain the progressive degeneration of the region to an uninterested Yelena. He breaks off, and she cross-examines him with regards to Sonya. It indeed turns out that Astrov does not love Sonya. He is, however, convinced of Yelena's own desire for him. Passionately he embraces her and insists upon arranging a rendezvous. Yelena momentarily relents; suddenly, however, Voynitsky enters, and she disengages herself from Astrov's arms. Voynitsky is quite disturbed.

Finally the other members of the household appear in the drawing room. Serebryakov announces that he plans to sell the estate. Voynitsky is livid, protesting that he has spent his best years working the land to the professor's benefit. Ominously Voynitsky storms out, and Yelena and Serebryakov both go after him. Suddenly a shot rings out off-stage. Serebryakov runs in; Voynitsky appears and fires a second shot. After a pause, it becomes clear he has missed twice. Dejected, Voynitsky tosses the revolver to the ground and sinks into a chair.

Act IV is set in Voynitsky's bedroom/estate office. Telegin and Marina sit winding stocking wool. Through their conversation, we learn that Yelena and the professor are departing that evening. Voynitsky and Astrov then enter, the latter asking Vanya to return a stolen bottle of morphine. Voynitsky declares that he is now nothing but a madman. For Astrov, Voynitsky is not mad but "eccentric"—such is the "normal condition" of man. Dreading the empty years to come, Voynitsky begs Astrov to help him start a new life. Annoyed, Astrov tells him he can do nothing: the provinces have poisoned them both. Sonya then enters, and after she pleads with him, Voynitsky surrenders the bottle.

Yelena appears and informs Voynitsky that her husband has sent for him. Begging her father to reconcile with the professor, Sonya exits with him. Yelena and Astrov say their subdued goodbyes. The doctor makes one more attempt to convince her to stay: Yelena declines. Astrov remarks that he is certain that had she remained, great "devastation" would ensue.

The other members of the household appear. Apparently Serebryakov and Voynitsky have reconciled. "Everyone will be just as it was," the latter murmurs grimly to the professor. Yelena and an apologetic Voynitsky share a brief farewell, Voynitsky telling her that she will never see him again. Terribly depressed, Voynitsky and Sonya return to their long-deferred work. Shortly thereafter Astrov departs as well.

Voynitsky then turns to Sonya, bemoaning his misery. Sonya tells him that they must endure their trials and wait for death. Laying her head on his lap, she conjures a vision of the heaven; her uncle weeps. The play closes with her repeated refrain "We shall rest we shall rest!"