Abstract: As a naturally occurring steroid sapogenin, diosgenin is an important intermediate for the synthesis of hundreds of steroid drugs. However, the biosynthetic pathway to diosgenin has not yet been completely elucidated and it remains unclear whether diosgenin biosynthesis originates from lanosterol or cycloartenol. In this study, the gene encoding cycloartenol synthase (CAS), TfCAS, was successfully cloned from a diosgenin-producing plant Trigonella foenum-graecum L. The open reading frame of the TfCAS gene had 2 271 base pairs of nucleotides encoding 756 amino acids. TfCAS showed 94%, 91%, and 89% of amino acid identities to the CASs from Medicago truncatula Gaertn., Pisum sativum L., and Lotus japonicas L., respectively. Biochemical analysis using a yeast expression system confirmed that TfCAS encodes a functional CAS protein that can convert 2,3-oxidosqualene into cycloartenol. The TfCAS transcript level in Trigonella foenum-graecum was further genetically modified by overexpressing the TfCAS gene via the hairy root transformation system. Real-time PCR analysis showed that the TfCAS transcript level was significantly up-regulated in the TfCAS-expressed hairy roots, suggesting success of the transgene events. Compared with the empty vector-transformed lines, the overexpression of TfCAS led to a slight but non-significant improvement in the biosynthesis of diosgenin and β-sitosterol. These data indicate that TfCASmay not be a rate limiting enzyme but may play a role in the diosgenin synthetic pathway in Trigonella foenum-graecum.