We demonstrated previously (S. Kawamura et al., Int. J. Cancer, 94: 343-347, 2001) that large amounts of ganglioside G(M3) accumulate in superficial bladder tumor, compared with invasive bladder tumors and that exogenous G(M3) inhibits the invasive potential of bladder tumor cells. To apply the G(M3) overexpression system to bladder tumor therapy, direct evidence for the important role of G(M3) in bladder tumor invasion must be obtained through transfer of the gene responsible for G(M3) overexpression. To determine the most appropriate cancer cell line for elucidating the antitumor effect of ganglioside G(M3) overexpression, the present study examined glycolipid composition, enzyme activity, and mRNA expression of the glycosyltransferases responsible for G(M3) synthesis in the bladder tumor cell lines KK-47, J82, MGH-UI, YTS-1, and MBT-2. A murine bladder carcinoma cell line (MBT-2) was transfected with a G(M3) synthase [(lactosylceramide alpha2,3-N-acetyl sialic acid transferase); sialyltransferase-I; SAT-I] cDNA, because this line does not naturally express G(M3). Stable transfectants (MBT-2-SAT-I) that overexpressed G(M3) were characterized by a reduced potential for cell proliferation, motility, invasion, and xenograft tumor growth, and an increase in the number of apoptotic cells. In the proportion of synthetic S phase, cells did not differ between MBT-2-SAT-I and mock-transfectant cells. These results suggest that the decreased proliferative potential related to G(M3) overexpression was attributable to the increased number of apoptotic cells. Although details of the mechanism of apoptosis remain unclear, the overexpression of G(M3) by gene transfer of SAT-I may present a novel therapeutic modality.