The structure of a cerebroside isolated from Hypsizigus marmoreus (mushroom, Bunashimeji) was determined to be (4E, 8E)-N-2-hydroxyhexadecanoyl-l-O-β-glucopyranosyl-9-methyl-C18-sphinga-4,8-dienine (1) by positive and negative ion mode fast atom bombardment (FAB) mass spectrometry using the B/E constant linked scan method. In particular, the location of the double bonds in the long-chain base was determined by the method. The structure of cerebrosides having the long-chain base of 9-methyl-C18-sphinga-4,8-dienine could be determined in general by the presence of characteristic fragment ions of [C19-sphingadienine + H - H2O]+ at m/z 276 and [C19 sphingadienine + H]+ at m/z 294, and the fatty acid carbon number could be calculated from the characteristic fragment ion of [ceramide - 18O]+ ([MH - GlcOH - 18O]+) in positive ion mode FAB mass spectrometry. Compound 1 was isolated for the first time from Hypsizigus marmoreus in the present study.