With an intense desire to serve the Lord burning within him, he attended a missionary conference being held in Manchester, and there he felt the divine call to carry the Gospel to the heathen. He was later accepted by the London Missionary Society, and at the age of 21, he sailed for Cape Town, South Africa. The hardships and primitive conditions did not deter him as he pushed northward into the interior, where he won to Christ the most dangerous outlaw chief in that region. 

Returning to Cape Town in 1819, he met his fiancée, arriving from England, and they were married. Together, they spent the next 51 years on the mission field, experiencing the many hardships and sorrows of that primitive area. Three of their children died in infancy and youth. However, five of the remaining ones remained in Africa as missionaries. Mary, the oldest daughter, became the wife of David Livingstone. The work of Moffat was, as it were, the stepping stones which others used in spreading the Gospel throughout the Dark Continent. He opened many mission stations and served as the pioneer missionary in an area of hundreds of square miles. He translated the Bible into the language of the Bechwanas, first having reduced the language to written characters. 

In 1870, after 54 years in Africa, he and his wife returned to England, where one year later she died. Moffat continued to promote foreign missions the rest of his life. He raised funds for a seminary that was built at the Kuruman Station, where native students were prepared for missionary work among their own people. At his death in 1883 the London newspaper said, "Perhaps no more genuine soul ever breathed. He addressed the cultured audiences within the majestic halls of Westminster Abbey with the same simple manner in which he led the worship in the huts of the savages."