Enantioselective epoxidation followed by regioselective epoxide opening reaction are the key processes in construction of the polyether skeleton. Recent genetic analysis of ionophore polyether biosynthetic gene dusters suggested that flavin-containing monooxygenases (FMOs) could be involved in the oxidation steps. In vivo and in vitro analyses of Lsd18, an FMO involved in the biosynthesis of polyether lasalocid, using simple olefin or truncated diene of a putative substrate as substrate mimics demonstrated that enantioselective epoxidation affords natural type mono- or bis-epoxide in a stepwise manner. These findings allow us to figure out enzymatic polyether construction in lasalocid biosynthesis.