Because of the poor response to chemotherapy and radiation therapy, new treatment approaches by immune-based therapy involving activated T cells are required for melanoma. We previously reported that the uncarboxylated form of osteocalcin (GluOC), derived from osteoblasts, potentially suppresses human prostate cancer cell proliferation by direct suppression of cell growth. However, the mechanisms in vivo have not been elucidated. In this study, we found that GluOC suppressed tumor growth of B16 mouse melanoma transplants in C57Bl/6N wild-type mice. Our data demonstrated that GluOC suppressed cell growth by downregulating phosphorylation levels of receptor tyrosine kinases and inducing apoptosis in vitro. Additionally, stimulation of primary mouse splenocytes with concanavalin A, a polyclonal T-cell mitogen, in the presence of GluOC increased T cell proliferation and their interferon-γ production. Taken together, we demonstrate that GluOC exerts multiple antitumor effects not only in vitro, but also in vivo through cellular immunostimulatory effects against B16 mouse melanoma cells.