- Horace Holly – protagonist and narrator, Holly is a Cambridge man whose keen intellect and knowledge were developed to compensate for his ape-like appearance. Holly knows a number of ancient languages, including Greek, Arabic, and Hebrew, which allow him to communicate with the Amahagger (who speak a form of Arabic) and She (who knows all three languages). Holly's interest in archaeology and the origins of civilisation lead him to explore the ruins of Kôr.
- Leo Vincey – ward of Horace Holly, Leo is an attractive, physically active young English gentleman with a head of thick blond hair. He is the confidant of Holly and befriends Ustane. According to She, Leo resembles Kallikrates in appearance and is his reincarnation.
- Ayesha – the title character of the novel, called Hiya by the native Amahagger, or "She" (She-who-must-be-obeyed). Ayesha was born over 2,000 years ago amongst the Arabs, mastering the lore of the ancients and becoming a great sorceress. Learning of the Pillar of Life in the African interior, she journeyed to the ruined kingdom of Kôr, feigning friendship with a hermit who was the keeper of the Flame that granted immortality. She bathed in the Pillar of Life's fire. Her name Ayesha is of Arabic origin and, according to the author, should be pronounced "Assha".[4]
- Job – Holly's trusted servant. Job is a working-class man and highly suspicious and judgmental of non-English peoples. He is also a devout Protestant. Of all the travellers, he is especially disgusted by the Amahagger and fearful of She.
- Billali – an aged elder of one of the Amahagger tribes, who develops a paternal bond with Holly that proves instrumental in the escape from the Amahagger by both Holly and Leo.
- Ustane – an Amahagger maiden. She becomes romantically attached to Leo, caring for him when he is injured, acting as his protector, and defying She to stay with him.
- Kallikrates – an ancient Greek, the husband of Amenartas, and ancestor of Leo. Two thousand years ago, he and Amenartas fled Egypt, seeking a haven in the African interior where they met Ayesha. There, She fell in love with him, promising to give him the secret of immortality if he would kill Amenartas. He refused, and, enraged, She struck him down.
- Amenartas – an ancient Egyptian priestess and ancestress of the Vincey family. As a priestess of Isis, she was protected from the power of She. When Ayesha slew Kallikrates, she expelled Amenartas from her realm. Amenartas gave birth to Kallikrates's son, beginning the line of the Vinceys (Leo's ancestors).
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