Objective The aim of this study is to investigate the effects of irradiation on the proliferation and differentiation of MC3T3-E1 osteoblastic cells.Methods MC3T3-E1 cells were irradiated 24 h after initial seeding. Gamma-radiation was administered at 0, 4, and 8 Gy as single doses by using a 60Co source. Cell proliferation was assessed at days 1, 3, 5, and 7 post-irradiation by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide assay. The collagen secretion of the cells was measured through sirius red staining at day 12 post-irradiation. The expressions of osteogenesis-related genes were assessed throughreal time fluorescence quantitative polymerase chain reaction at day 16 post-irradiation. The matrixminera-lization caused by cells was evaluated through alizarin red staining at day 28 post-irradiation.Results The cells exposed to 4 Gy or 8 Gy demonstrated significantly lower proliferation rates compared with the non-irradiated group. Doses of 4 Gy or more significantly inhibited the expressions of osteogenesis-related genes(Osterix and osteocalcin). Collagen secretion and cell mineralization were significantly reduced by the 8 Gy dose.Conclusion 60Co γ-rays dose-dependently suppress the proliferation, collagen secretion, and mineralization of MC3T3-E1 cells. Furthermore, radiation seems to dose-dependently inhibit the expressions of osteogenesis-related genes of the cells.