Sterols are isoprenoid-derived lipids that are produced via the mevalonate pathway and are involved in various cellular functions in eukaryotes such as maintenance of membrane integrity and biosynthetic precursors of steroid hormones. Among cellular sterols, Delta(22)-sterols containing a double bond at C-22 in the sterol side chain specifically occur in fungi (ergosterol) and plants (stigmasterol and brassicasterol), and several lines of experimental evidence have suggested specific physiological roles of Delta(22)-sterols in plants. Fungal cytochrome P450 (P450), CYP61, has been established as the sterol C-22 desaturase functioning at the penultimate step in the ergosterol biosynthetic pathway. On the other hand, no particular sequence has been assigned as to the enzyme responsible for the introduction of the double bond into the sterol side chain in plants. In this review, we summarize our recent findings demonstrating that CYP710A P450 family genes encode the plant sterol C-22 desaturases to produce stigmasterol and brassicasterol/crinosterol from beta-sitosterol and 24-epi-campesterol respectively.