Maesasaponins produced by the African shrub Maesa lanceolata are oleanane-type saponins with diverse biological activities. Candidate maesasaponin biosynthesis genes were identified through transcript profiling of M. lanceolata shoot cultures treated with methyl jasmonate, a well-known elicitor of plant specialized metabolism. Heterologous expression of the identified genes in Saccharomyces cerevisiae led to the identification of one oxidosqualene cyclase (MlbAS) and two cytochrome P450s, CYP716A75 and CYP87D16, which catalyse three enzymatic steps of maesasaponin biosynthesis. MlbAS is a β-amyrin synthase that converts 2,3-oxidosqualene to β-amyrin in yeast, and its role in maesasaponin biosynthesis was confirmed by gain- and loss-of-gene-function experiments in transgenic M. lanceolata plants. When expressed in β-amyrin-producing yeast, CYP716A75 catalyses the C-28 oxidation of β-amyrin, leading to the accumulation of mainly erythrodiol. Accordingly, expression of CYP716A75 in a Medicago truncatula mutant lacking C-28 oxidase activity partially complemented the mutant phenotype. CYP87D16 catalyses the C-16α oxidation of β-amyrin in yeast, leading to the accumulation of 16α-hydroxy β-amyrin. This activity was hitherto only linked to a CYP716-type P450 from a distinct plant species, thereby suggesting that C-16α oxidase activity has evolved independently in different plant lineages. The identified biosynthesis genes will be useful for synthetic biology programs toward the production of bioactive triterpenoids.