Injun Joe stands as the narrative's primary antagonist, shaped by social exclusion and mistreatment that fuel his motivations. He references being denied food by Dr. Robinson's father and subsequent imprisonment for vagrancy, sparking his vendetta. He attributes his vengeful nature to his partial Native American heritage, declaring, "The Injun blood ain't in me for nothing."

His pivotal actions commence with grave robbing alongside Dr. Robinson and Muff Potter. During this encounter, he murders Dr. Robinson and frames the intoxicated Potter for the crime. Later, he plots to mutilate Widow Douglas in retaliation for her husband—a justice of the peace—having him publicly whipped.

Joe demonstrates cunning adaptability, evading capture by disguising himself as a deaf-mute Spaniard. His pursuit of treasure drives a key plot thread, culminating in the concealment of a significant hoard of gold coins.

His interactions with Tom Sawyer and Huckleberry Finn are defined by mutual dread. The boys witness the graveyard murder but remain silent under a blood oath, fearing his retribution. Joe later learns of their knowledge and threatens Tom’s life. During the murder trial, Tom’s testimony exposes Joe’s guilt, forcing his escape.

Trapped in McDougal’s Cave after its entrance is sealed, Joe perishes near the barrier. His death results from starvation, his final moments fixated on the world beyond. Tom and Huck later discover his hidden gold.

Across adaptations like the 1980 anime series and 1995 live-action film, his core characterization persists. The film introduces minor variations, including a treasure map subplot and an altered death scene involving a fatal fall during pursuit. He remains a vengeful, static figure until his demise in all official media.