Triterpenoids are a large and medicinally important group of natural products with a wide range of biological and pharmacological effects. Among them, malabaricane-type triterpenoids are a rare group of terpenoids with a 6,6,5-tricyclic ring system, and a few malabaricane triterpene synthases have been characterized to date. Here, an arabidiol synthase AmAS for the formation of the malabaricane-type 6,6,5-tricyclic triterpenoid skeleton in astramalabaricosides biosynthesis was characterized from Astragalus membranaceus. Multiple sequence alignment, site-directed mutagenesis, and molecular docking of AmAS reveal that residues Q256 and Y258 are essential for AmAS activity, and the triad motif IIH725-727 was the critical residue necessary for its product specificity. Mutation of IIH725-727 with VFN led to the formation of seven tricyclic, tetracyclic, and pentacyclic triterpenoids (1-7). Glycosylation of malabaricane-type triterpenoids in the biosynthesis of astramalabaricosides was also explored. Three triterpenoids (1, 5, and 6) displayed potent inhibitory effects against influenza A virus in vitro. These findings provide insights into malabaricane-type triterpenoids biosynthesis in A. membranaceus and access to diverse bioactive triterpenoids for drug discovery.