T antigen (Galβ1-3GalNAcα1-Ser/Thr), a core 1 mucin-type O-glycan structure, is synthesized by Drosophila core 1 β1,3-galactosyltrasferase 1 (dC1GalT1) and is expressed in various tissues. We previously reported that dC1GalT1 synthesizes T antigen expressed in hemocytes, lymph glands, and the central nervous system (CNS) and that dC1GalT1 mutant larvae display decreased numbers of circulating hemocytes and excessive differentiation of hematopoietic stem cells in lymph glands. dC1GalT1 mutant larvae have also been shown to have morphological defects in the CNS. However, the functions of T antigen in other tissues remain largely unknown. In this study, we found that glycans contributed to the localization of neuromuscular junction (NMJ) boutons. In dC1GalT1 mutant larvae, NMJs were ectopically formed in the cleft between muscles 6 and 7 and connected with these two muscles. dC1GalT1 synthesized T antigen, which was expressed at NMJs. In addition, we determined the function of mucin-type O-glycans in muscle cells. In dC1GalT1 mutant muscles, myofibers and basement membranes were disorganized. Moreover, ultrastructural defects in NMJs and accumulation of large endosome-like structures within both NMJ boutons and muscle cells were observed in dC1GalT1 mutants. Taken together, these results demonstrated that mucin-type O-glycans synthesized by dC1GalT1 were involved in the localization of NMJ boutons, synaptogenesis of NMJs, establishment of muscle cell architecture, and endocytosis.