Abstract: In order to study the biosynthesis mechanism, a lanosterol synthase gene (AcLSS) from Antrodia cinnamomea was isolated and cloned in this study, and then the physicochemical properties were predicted by using bioinformatics software, and the gene expressions were also analyzed. The results showed that the open reading frame of AcLSS gene was 2205 bp, which encoding 734 amino acids; AcLSS gene had 8 introns and 9 exons; AcLSS protein had conserved characteristic sequences which consist of 14 amino acids 'KGAWPFSTKTQGYT' and 6 amino acids 'VSDCTG', as well as a OSC conserved domain which consist of 16 amino acids 'KACNFLISKQRSDGGW'. The molecular phylogenetic analysis revealed that AcLSS protein was most closely related to the LSS protein of Sparassis crispa (XP_027617410.1). The gene expression profile indicated that the best ability of induction expression AcLSS gene was the mannitol from carbon source and the tomato powder from nitrogen source, respectively.