Melanoma is the most malignant form of skin cancer, which originates from the pigment-producing melanocytes in the basal layer of epidermis. L-type amino acid transporter 1 (LAT1) is an amino acid transporter that transports most of the essential amino acids. LAT1 is upregulated in various cancers, including melanoma, and contributes to supporting cancer cell proliferation. Therefore, LAT1 is regarded as a promising molecular target of novel anti-cancer therapeutics. In this study, we established a syngeneic and orthotropic mouse model for melanoma to examine the anti-tumor effects of LAT1 inhibitor. B16F10 mouse melanoma cells were orthotopically injected into footpads of C57BL/6J mice. One week after the inoculation, intravenous injection of LAT1 inhibitor was started and continued once-daily for two weeks. The tumor size on footpads of LAT1 inhibitor-treated mice was significantly reduced compared to those of saline-treated control mice, demonstrating the anti-tumor effects of LAT1 inhibitor. Our syngeneic and orthotropic model will be useful to evaluate the therapeutic efficacy of LAT1 inhibitors for melanoma in the presence of intact immune system, and also to investigate its detailed mechanisms of action as anti-tumor therapeutics.