Cholesterol is the precursor of bioactive plant metabolites such as steroidal saponins. An Australian plant,Dioscorea transversa, produces only two steroidal saponins: 1β-hydroxyprotoneogracillin and protoneogracillin. Here, we usedD. transversaas a model in which to elucidate the biosynthetic pathway to cholesterol, a precursor to these compounds. Preliminary transcriptomes ofD. transversarhizome and leaves were constructed, annotated, and analyzed. We identified a novel sterol side-chain reductase as a key initiator of cholesterol biosynthesis in this plant. By complementation in yeast, we determine that this sterol side-chain reductase reduces Δ24,28double bonds required for phytosterol biogenesis as well as Δ24,25double bonds. The latter function is believed to initiate cholesterogenesis by reducing cycloartenol to cycloartanol. Through heterologous expression, purification, and enzymatic reconstitution, we also demonstrate that theD. transversasterol demethylase (CYP51) effectively demethylates obtusifoliol, an intermediate of phytosterol biosynthesis and 4-desmethyl-24,25-dihydrolanosterol, a postulated downstream intermediate of cholesterol biosynthesis. In summary, we investigated specific steps of the cholesterol biosynthetic pathway, providing further insight into the downstream production of bioactive steroidal saponin metabolites.