John Brownlee was originally trained as a gardener, but then studied theology at Glasgow University. He was ordained in September 1816 and sent to South Africa by the London Missionary Society, arriving in April 1817 on the same ship as Robert Moffat. He married in 1818 and in the same year resigned from the London Missionary Society. In 1820 he accepted an appointment by Lord Charles Somerset, governor of the Cape Colony, as a missionary and government agent among Chief Gaika's people in Kaffraria. He stayed mainly at the Tyumi Mission Station near present day Alice, before rejoining the London Missionary Society in 1825 and founding a mission station at the site where King William's Town now stands. Though this mission was sacked during the Frontier Wars of 1834-1835 and 1846-1847 he worked there till his retirement in 1867. Interested in politics, literature and theology, he is particularly known as a Xhosa linguist. He wrote an "Account of the Amakosae, or Southern Caffres" that was published in Travels and Adventures in Southern Africa (1827) by George Thompson*.
Brownlee was an enthusiastic plant collector and had an extensive knowledge of the local flora. During the eighteen-sixties the Colonial Botanist John C. Brown* encouraged him to send in specimens, most of which were passed on to W.H. Harvey* in Dublin. He also sent plants to Kew Gardens. Harvey named a genus of mostly South African ground orchids in his honour. The first two species in this genus, Brownleea caerulea and Brownleea parviflora, were discovered by Brownlee near his mission station. As an active gardener he furthermore introduced many valuable cultivated plants at King William's Town.
Brownlee's wife, Maria Brownlee, was killed during the frontier war of 1850-1853. He subsequently married Catharina de Jager.