Riding on a train through England, Justice Wargrave begins thinking about where he is going: Soldier Island. He remembers a story of a millionaire who lived there, but has heard that the Island was recently bought by a man named Mr. Owen. The scene quickly changes to another woman on the train, Vera Claythorne who is similarly curious about this island. The narrative shifts among a variety of other characters, the mysterious Philip Lombard, the rigid spinster Miss Emily Brent, the retired General Macarthur, the successful Dr. Armstrong, the reckless Anthony Marston, and the lying Mr. Blore, who are all also headed to Soldier Island.
They are taken by boat to the island by a local named Fred Narracott. At the island the host is not there but two servants, Mr. and Mrs. Rogers, tell the guests that dinner is almost ready. The guests go up to their rooms and notice that there is a nursery rhyme hung in each, “Ten Little Soldiers.” In each verse another Little Soldier dies in some strange way. After a pleasant dinner, the guests all gather in the parlor for drinks. All of a sudden a loud voice takes over the room, accusing each guest, one at a time, of murder. When the recording ends, Mrs. Rogers faints and is taken upstairs by her husband and the doctor.
The voice has come from a gramophone hidden in the next room. Justice Wargrave, a retired judge, begins an impromptu court session – asking everyone to explain his or her accusation. Each guest has some sort of excuse. They realize that they have all been invited to the island under false pretenses by someone with the name U. N. Owen which, as Wargrave points out, spells out Unknown. Then, all of a sudden, Anthony Marston chokes, falls over, and dies. Dr. Armstrong checks Marston's drink and discovers that it has been poisoned! They take him up to his room and all decide that they will try to leave in the morning. In bed in her room Vera realizes that Marston's death mirrors the first line of the Ten Little Soldiers rhyme.
The next morning Mr. Rogers tells everyone that Mrs. Rogers died in the middle of the night. He also says that two of the little soldier figurines that had been decorating the table had now disappeared. The guests now decide that there must be a “raving maniac” on the island. They all think that they should leave but a storm is coming and no boat will be able to get to or from the island. Blore and Lombard decide to search the island but find no one. They do discover that Lombard has a revolver. Mr. Rogers calls everyone in for lunch, but they realize that General Macarthur is not there. Dr. Armstrong goes to check on him and finds him dead – hit on the head with a life preserver. Wargrave holds another court-session and decides that although no one person stands out as the murderer, the killer must be one of the guests on the Island.
The next morning they wake to find Rogers dead – struck on the back of head with an axe while chopping wood. Vera and Miss Brent prepare breakfast now that there are no surviving servants. After breakfast, Miss Brent feels weak so stays seated as the rest of the guests clear the table. When they come back they find Miss Brent dead. She has been poisoned with a syringe in the neck and the murderer even took the time to put a bee in the room (to match the verse in the Ten Little Soldiers rhyme). Wargrave suggests that they should lock up any dangerous items that they have brought, but when Lombard goes to get his revolver he realizes that it is missing.
Next Wargrave dies, shot through the head and dressed up as a judge while a decoy of a drowning scene frightens Vera and distracts all the other guests. That night Lombard hears a noise from his room and wakes up everyone else only to realize that Dr. Armstrong is missing. They believe that Armstrong must be the murderer. They spend the morning outside the house where it feels open and safe. Finally Blore decides to get some food from the house, but on his way inside he is crushed by a bear shaped statue dropped from above and which had been sitting on Vera's mantelpiece. Lombard decides that they should find a high place on the Island and stay awake all night but when they get there they find Armstrong dead. Vera and Lombard are the only two remaining on the island. They both suspect the other but Vera manages to snatch Lombard's revolver and shoot him. She then feels a sudden calm and walks back to the house intending to eat something and sleep. Instead she thinks about the little boy she tricked into drowning in an attempt to free the man she loved to marry her (except that the man figured it out and left her immediately). She notices a noose hanging in her bedroom and a chair placed just under it, and hangs herself.
The epilogue is set in Scotland Yard where Sir Thomas Legge and Inspector Maine are discussing the mystery murders on Soldier Island. They go through all the facts but cannot understand how everyone on the Island could be dead. The final chapter is a letter from Wargrave describing how he has always been obsessed with justice and death. As a small child he enjoyed killing animals, but he never wanted to harm anything innocent. This led him to a career in the law, but he always felt something lacking. After learning he was soon to die from a medical condition, he decided that for his final act in life he wanted to commit the perfect murder. But he also did not want to kill innocent people, so he collected a group of men and women who had committed crimes for which they had never been punished, and brought them to Soldier Island. Then he killed them one by one, saving the guiltiest for last. He hid his own involvement by getting the doctor to help fake his own death (tricking the doctor into thinking such a ruse would help out the “real” murderer), and then killing the doctor. After Vera hanged herself, Wargrave arranged the house just so, and then carefully killed himself.