Yessotoxin (YTX) is a ladder-frame polyether produced by the marine dinoflagellate Protoceratium reticulatum. Biosynthetic studies on ladder-frame polyethers have lagged behind their structural elucidation and synthetic studies, and been limited to ^<13>C-incorporation patterns to their carbon backbones. In addition to the origin of carbon atoms, origins of the oxygen atoms are also much intriguing for elucidation of formation of ether rings and hydroxyls. Although the ladder-frame polyethers have been hypothesized to be produced through a polyene precursor followed by epoxidation and cascade cyclization to give the ladder-like structures, no experimental evidence has been reported on their biosynthesis. We investigated biosynthesis of YTX by feeding ^<18>O-labeled precursors into the cultured producer to identify the origins of the oxygen atoms using collision-induced dissociation tandem mass spectrometry (CID-MS/MS). The CID-MS/MS analyses demonstrated that all the ether oxygens and the sulfate ester oxygen on C4 were derived from molecular oxygen, and the hydroxyl on C32 originated from acetate carbonyl, supported by ^<13>C-acetate incorporation pattern of YTX. These results also provided us with new insights about construction of the skeletal structure. Besides revealing the origin of the oxygen atoms, we conducted a feeding experiment with [2-1^<13>CD_3]acetate into YTX to confirm mechanism of branched methylation from acetate methyl by ^<13>C NMR. The D-decoupled ^<13>C NMR spectrum gave D_2-retained signals at pendant methyl carbons, indicating an acetyl-CoA directly condensed with an oxidized 13-carbon along elongation of the carbon chain.