Sineoculis homeobox homolog 1 (Six1) is one of the transcription factors that act as master regulators of development and is frequently dysregulated in cancers. However, the biological role of Six1 is not clear in osteosarcoma. To address the expression of Six1 in osteosarcoma cells, three osteosarcoma cell lines (U2OS, SaOS-2, and MG63) and a human osteoblastic cell line (hFOB1.19) were used to detect the expression of Six1 by quantitative real-time polymerase chain reaction and western blotting. The results showed that Six1 was upregulated in osteosarcoma cell lines compared to human osteoblastic cell line hFOB1.19. To investigate the role of Six1 in osteosarcoma cells, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, flow cytometry analysis, and transwell chamber assays were used to determine the effects of Six1 on the cell viability, cycle, apoptosis, and migration properties in U2OS cells. The results showed that Six1 could promote U2OS cell proliferation and migration, and suppress U2OS cell apoptosis. In addition, we investigated the effects of Six1 on the expression of following proteins (cyclin D1, caspase-3, and vascular endothelial growth factor-C (VEGF-C)). Results showed that Six1 could increase the expression of cyclin D1 and VEGF-C, and decrease the expression of caspase-3. All these data suggested that Six1 might be involved in the promotion of growth, proliferation, and migration of U2OS cells, as well as the inhibition of apoptosis of U2OS cells. These data might provide information for the prediction of osteosarcoma prognosis and potential targets for therapy of osteosarcoma.