Histone deacetylase inhibitors such as butyrate are known to exhibit anti-cancer activities in a wide range of cancer including melanoma. In spite of these potencies, butyrate is not practically used for cancer treatment due to its rapid metabolism and very short plasma half-life. Tributyrin, a triglyceride analog of butyrate, can act as a pro-drug of butyrate after being cleaved by intracellular enzymes. The present study sought to investigate a possibility to develop tributyrin emulsion as a potent anti-cancer agent against melanoma. Mixture of Tween80 and 1, 2-dimyristoyl-sn-glycero-3-phosphocholine as a surfactant to disperse tributyrin produced homogeneous emulsions with nanometer sizes, even without a harsh homogenization procedure. Tributyrin emulsion was more potent than butyrate in inhibiting the growth of B16-F10 melanoma cells. Accumulation of cells at sub G(0)/G(1) phase and the DNA fragmentation induced by tributyrin emulsion treatment revealed that tributyrin emulsion inhibited the growth of B16-F10 cells by inducing apoptosis. Treatment with tributyrin emulsion suppressed the colony formation of melanoma cells in a dose-dependent manner. Furthermore, after intraperitoneal administration into mice, tributyrin emulsion inhibited the formation of tumor colonies in the lung following intravenous injection of melanoma cells. Taken together, our data suggests that tributyrin emulsion may be developed as a potent anti-cancer agent against melanoma.