Ginkgolides and bilobalide, collectively termed terpene trilactones (TTLs), are terpenoids that form the main active substance of Ginkgo biloba. Terpenoids in the mevalonate (MVA) biosynthetic pathway include acetyl-CoA C-acetyltransferase (AACT) and mevalonate kinase (MVK) as core enzymes. In this study, two full-length (cDNAs) encoding AACT (GbAACT, GenBank Accession No. KX904942) and MVK (GbMVK, GenBank Accession No. KX904944) were cloned from G. biloba. The deduced GbAACT and GbMVK proteins contain 404 and 396 amino acids with the corresponding open-reading frame (ORF) sizes of 1215 bp and 1194 bp, respectively. Tissue expression pattern analysis revealed that GbAACT was highly expressed in ginkgo fruits and leaves, and GbMVK was highly expressed in leaves and roots. The functional complementation of GbAACT in AACT-deficient Saccharomyces cerevisiae strain Δerg10 and GbMVK in MVK-deficient strain Δerg12 confirmed that GbAACT mediated the conversion of mevalonate acetyl-CoA to acetoacetyl-CoA and GbMVK mediated the conversion of mevalonate to mevalonate phosphate. This observation indicated that GbAACT and GbMVK are functional genes in the cytosolic mevalonate (MVA) biosynthesis pathway. After G. biloba seedlings were treated with methyl jasmonate and salicylic acid, the expression levels of GbAACT and GbMVK increased, and TTL production was enhanced. The cloning, characterization, expression and functional analysis of GbAACT and GbMVK will be helpful to understand more about the role of these two genes involved in TTL biosynthesis.