Between the early 16th and late 19th centuries, an estimated 12 million Africans were transported to the Americas as part of the transatlantic slave trade. Following Britain’s abolition of slave trade in 1807, the Royal Navy patrolled the Atlantic and intercepted slave ships that continued to operate. During this period, the island of St Helena in the middle of the South Atlantic served as a depot for “liberated” Africans. Between 1840 and 1867, approximately 27,000 Africans were disembarked on the island. To investigate their origins, we generated genome-wide ancient DNA data for 20 individuals recovered from St Helena. The genetic data indicate that they came from West Central Africa, possibly the area of present-day Gabon and Angola. The data further suggest that they did not belong to a single population, confirming historical reports of cultural heterogeneity among the island’s African community. Our results shed new light on the origins of enslaved Africans during the final stages of the slave trade and illustrate how genetic data can be used to complement and validate existing historical sources.