- The novel's protagonist, the Lady of the title. Isabel is a young woman from Albany, New York, who travels to Europe with her aunt, Mrs. Touchett. Isabel's experiences in Europe—she is wooed by an English lord, inherits a fortune, and falls prey to a villainous scheme to marry her to the sinister Gilbert Osmond—force her to confront the conflict between her desire for personal independence and her commitment to social propriety. Isabel is the main focus of Portrait of a Lady,
and most of the thematic exploration of the novel occurs through her actions, thoughts, and experiences. Ultimately, Isabel chooses to remain in her miserable marriage to Osmond rather than to violate custom by leaving him and searching for a happier life.
- A cruel, narcissistic gentleman of no particular social standing or wealth, who seduces Isabel and marries her for her money. An art collector, Osmond poses as a disinterested aesthete, but in reality he is desperate for the recognition and admiration of those around him. He treats everyone who loves him as simply an object to be used to fulfill his desires; he bases his daughter Pansy's upbringing on the idea that she should be unswervingly subservient to him, and he even treats his longtime lover Madame Merle as a mere tool. Isabel's marriage to Osmond forces her to confront the conflict between her desire for independence and the painful social proprieties that force her to remain in her marriage.
- An accomplished, graceful, and manipulative woman, Madame Merle is a popular lady who does not have a husband or a fortune. Motivated by her love for Gilbert Osmond, Merle manipulates Isabel into marrying Osmond, delivering Isabel's fortune into his hands and ruining Isabel's life in the process. Unbeknownst to either Isabel or Pansy, Merle is not only Osmond's lover, but she is also Pansy's mother, a fact that was covered up after Pansy's birth. Pansy was raised to believe that her mother died in childbirth.
- Isabel's wise, funny cousin, who is ill with lung disease throughout the entire novel, which ends shortly after his death. Ralph loves life, but he is kept from participating in it vigorously by his ailment; as a result, he acts as a dedicated spectator, resolving to live vicariously through his beloved cousin Isabel. It is Ralph who convinces Mr. Touchett to leave Isabel her fortune, and it is Ralph who is the staunchest advocate of Isabel remaining independent. Ralph serves as the moral center of Portrait of a Lady
: his opinions about other characters are always accurate, and he serves as a kind of moral barometer for the reader, who can tell immediately whether a character is good or evil by Ralph's response to that character.
- An aristocratic neighbor of the Touchetts who falls in love with Isabel during her first visit to Gardencourt. Warburton remains in love with Isabel even after she rejects his proposal and later tries to marry Pansy simply to bring himself closer to Isabel's life.
- The son of a prominent Boston mill owner, Isabel's most dedicated suitor in America. Goodwood's charisma, simplicity, capability, and lack of sophistication make him the book's purest symbol of James's conception of America.
- Isabel's fiercely independent friend, a feminist journalist who does not believe that women need men in order to be happy. Like Caspar, Henrietta is a symbol of America's democratic values throughout he book. After Isabel leaves for Europe, Henrietta fights a losing battle to keep her true to her American outlook, constantly encouraging her to marry Caspar Goodwood. At the end of the book, Henrietta disappoints Isabel by giving up her independence in order to marry Mr. Bantling.
- Isabel's aunt. Mrs. Touchett is an indomitable, independent old woman who first brings Isabel to Europe. The wife of Mr. Touchett and the mother of Ralph, Mrs. Touchett is separated from her husband, residing in Florence while he stays at Gardencourt. After Isabel inherits her fortune and falls under the sway of Merle and Osmond, Mrs. Touchett's importance in her life gradually declines.
- Gilbert Osmond's placid, submissive daughter, raised in a convent to guarantee her obedience and docility. Pansy believes that her mother died in childbirth; in reality, her mother is Osmond's longtime lover, Madame Merle. When Isabel becomes Pansy's stepmother, she learns to love the girl; Pansy is a large part of the reason why Isabel chooses to return to Rome at the end of the novel, when she could escape her miserable marriage by remaining in England.
- A hapless American art collector who lives in Paris, Rosier falls in love with Pansy Osmond and does his best to win Osmond's permission to marry her. But though he sells his art collection and appeals to Madame Merle, Isabel, and the Countess Gemini, Rosier is unable to change Gilbert's mind that Pansy should marry a high-born, wealthy nobleman, not an obscure American with little money and no social standing to speak of.
- An elderly American banker who has made his life and his vast fortune in England who is Ralph's father and the proprietor of Gardencourt. Before Mr. Touchett dies, Ralph convinces him to leave half his fortune to his niece Isabel, which will enable her to preserve her independence and avoid having to marry for money.
- The game Englishman who acts as Henrietta's escort across Europe, eventually persuading her to marry him at the end of the novel.
- Osmond's vapid sister, who covers up her own marital infidelities by gossipping constantly about the affairs of other married women. The Countess seems to have a good heart, however, opposing Merle's scheme to marry Osmond and Isabel and eventually revealing to Isabel the truth of Merle's relationship to Osmond and Pansy's parentage.