The form of selenium appears to be important for preventing cancer in humans. Here, we evaluated selenium levels in the serum and bone tissue samples from osteosarcoma patients using atomic absorption spectrometry. The in vitro effects of Se-methylselenocysteine (Se-MSC) on growth, cell cycle status, and apoptosis of osteosarcoma cells were assessed using the WST-1 assay, Hoechst 33342/propidium iodide staining, and flow cytometry, respectively. In osteosarcoma cases, the mean serum selenium levels in osteosarcoma tissue and normal bone were 0.08 mg/kg and 0.03 mg/kg, respectively (P < 0.05). Serum selenium levels in osteosarcoma and non-osteosarcoma cases were 0.09 mg/L and 0.08 mg/L, respectively (P > 0.05). Se-MSC-treated MG63 cells showed altered cellular morphology, decreased viability in a time- and dose-dependent manner, and an increase in the sub-G1 cell population. Se-MSC also downregulated Bcl-2 expression and upregulated Bax. Se-MSC inhibited the proliferation of the drug-resistant osteosarcoma cell line Saos-2/MTX300 and enhanced the inhibitory effect of pirarubicin on MG63 cells. Our data demonstrate that selenium levels are significantly higher in osteosarcoma tissue than normal bone tissue in osteosarcoma patients. The results also support the anticancer effects of Se-MSC in osteosarcoma. Further development of Se-MSC as an ancillary chemotherapy agent in osteosarcoma is warranted.