Liquid chromatography-mass spectrometry is one of the most powerful methods for the identification and detection of chemical structures of lipids. In this study, we attempted to identify the chemical structures of glucosylceramides from maize, rice, mushroom (maitake) and sea cucumber by liquid chromatography-ion trap mass spectrometry. For structural analysis of glucosylceramides, [M + H](+),[M + H - 18](+) or [M + H - 162](+) in the positive scan mode was used for MS/MS analysis to obtain product ion spectra. The typical signals which are characteristic for the sphingoid base moieties were observed while the isomers could not be distinguished. This method should be useful for the structural determination of diverse glucosylceramide molecular species.