Cancer vaccination elicits an immune response against specific glycans or proteins expressed on the cell surface after gene transfer has occurred. We previously demonstrated that N-acetylglucosamine (GlcNAc) residues exposed after digestion with endo-?-galactosidase, a carbohydrate-digesting enzyme, elicited this type of immune response, probably as a result of the presence of natural antibodies recognizing GlcNAc residues in host animals. Treatment of a cell with endo-?-N-acetylglucosaminidase (ENGase), an enzyme that cleaves the amide bond between the proximal GlcNAc residues at the side chain of an asparagine residue on N-glycans, also causes exposure of GlcNAc residues on the cell surface. In this study, we examined whether mouse melanoma B16 cells transfected with a human ENGase (hENGase) cDNA expression construct, are susceptible to an immune attack after subcutaneous grafting to the syngenic host. The recombinant B16 cells overexpressing hENGase had approximately 3-fold more cell-surface GlcNAc residues than their parental cells. The grafting experiment revealed that the tumor size was approximately one-tenth of that derived from wild-type grafted cells. Direct injection and subsequent in vivo electroporation of a hENGase expression vector into B16 solid tumors resulted in regression of the tumors. Our present results strongly suggest that the ENGase is a useful tool for novel cancer vaccination.