With little training, Moffat was assigned in 1816 by the London Missionary Society to go to South Africa. After spending seven years in several locations that were disrupted by warfare among Zulu tribesmen, he settled at Kuruman, southeast of the Kalahari (desert). There he lived for 49 years, building one of the foremost Protestant missionary communities in Africa. He traveled widely, and he encountered numerous tribes and mastered the Tswana language, into which he translated the Gospel According to Luke (1830). Through his influence the number of converts rose rapidly, and by 1857 he had completed a Tswana translation of the entire Bible. In 1838 he wrote A Book of Hymns in Chuana (Tswana).

Though criticized by some as paternalistic, he laboured not only to alter the conduct but also to raise the standard of living of African peoples by introducing improved methods of agriculture and irrigation. On his first meeting with Livingstone, in 1840, Moffat recognized Livingstone's capacities and urged him to come to Africa, directing him to the region north of the Kalahari. After the death of his wife, Mary, with whom he shared his work for half a century, Moffat lived his remaining 13 years in retirement in England.

Among Moffat's writings are Missionary Labours and Scenes in Southern Africa (1842) and Rivers of Water in a Dry Place (1863).