Osteoblasts are bone-forming cells that are responsible for the production of bone extracellular matrix. Osteoradionecrosis is a complication of radiation therapy for carcinoma of the head and neck that occurs in 3% to 8.2% of irradiated patients. The irradiation effect on osteoblast differentiation has not been fully elucidated. The objective of our research was to elucidate the effects of radiation on the growth and differentiation-related gene expression in osteoblast in vitro. Three differentiation-related genes, alkaline phosphatase, type I collagen, and osteocalcin, were tested in our experiment. The results showed that radiation inhibited the proliferation of the osteoblasts in a dose-dependent manner and induced G2/M cell cycle arrest. Irradiation, 4 and 8 Gy, enhanced the differentiation-related gene expression of MC3T3-E1 cells 7 days after irradiation. However, the differentiation-related gene expression was decreased 21 days after irradiation in the 4- and 8-Gy groups. This work presents the dynamic phenotypic expression changes of osteoblastic cells after x-ray irradiation.