This study describes the structure-cytotoxicity relationship of beta-glucosylceramide (beta-GluCer) and its antitumor activity in vivo. Unglycosylated ceramide had no selective cytotoxicity which demonstrated that the sugar moiety plays a critical role for the expression of selective cytotoxicity by beta-GluCer. beta-Galactosylceramide also showed tumor specific cytotoxicity suggesting that the chemical structure of sugar group is not a factor for the selective toxicity. Similarly, unglycosylated ceramides of short acyl chain also selectively inhibited the growth of cancer cells. These findings in concert point to the importance of the hydrophilicity of the ceramide molecule rather than the chemical structure for the cyto-selectivity. Treatment of the cells with beta-GluCer increased the concentration of reactive oxygen species leading to cell cycle arrest and necrosis. Intraperitoneal administration of beta-GluCer significantly suppressed the growth of tumor implanted to the back of mice. beta-GluCer also induced antitumor immunity via the activation of NKT cells in vivo, and decreased the tumor metastasis of lymphoma cells. The present study thus demonstrated the antitumor activity of beta-GluCer in vivo, and discussed the mechanisms responsible for the growth inhibition.