Glycosphingolipids (GSLs) from the silkworm Bombyx mori were identified and GSL expression patterns between larvae and pupae were compared. The structural analysis of neutral GSLs from dried pupae revealed the following predominant species: Glcβ1Cer, Manβ4Glcβ1Cer, GlcNAcβ3Manβ4Glcβ1Cer, Galβ3Manβ4Glcβ1Cer, GalNAcα4Galβ3Manβ4Glcβ1Cer, GlcNAcβ3Galβ3Manβ4Glcβ1Cer, Galα4Galβ3Manβ4Glcβ1Cer and (GalNAcα4)1-4 GalNAcα4Galβ3Manβ4Glcβ1Cer. Lin-ear elongation of α4-GalNAc was observed at the non-reducing end of Galβ3Manβ4Glcβ1Cer with up to five GalNAc repeats. The arthro-series GSL GlcNAcβ3Manβ4Glcβ1Cer, a characteristic GSL-glycan sequence of other Arthropoda, was detected in silkworms. The main ceramide species in each purified GSL fraction were h20:0-d14:1 and h22:0-d14:1. GSL expression patterns in larvae and pupae were compared using thin-layer chromatography, which demonstrated differences among acidic, polar and neutral GSL fractions, while the zwitterionic fraction showed no difference. Neutral GSLs such as ceramides di-, tri- and tetrasaccharides in larvae showed less abundant than those in pupae. MALDI-TOF MS analysis revealed that larval GSLs contained four types of ceramide species, whereas pupal GSLs contained only two types. The structural analysis of neutral GSLs from silkworms revealed a novel series of GSLs. The comparison of GSL expression patterns between larvae and pupae demonstrated differences in several fractions. Alterations in GSL ceramide composition between larvae and pupae were observed by MALDI-TOF MS analysis.