Background and Objective Bone injury is a common side effect of radiotherapy to tumors, which is a long-term response after damage to osteoblasts, especially reducing osteoblasts proliferation and differentiation. Currently, there are few studies on radiation-induced bone injury, the molecules involved in the ionizing radiation (IR) induced osteoblasts damage remain need to be excavated. Therefore, this study aims to establish a radiation induced osteoblast injury model, to screen and identify relevant factors involved in radiation injury of osteoblast through RNA-sequencing. Methods The MC3T3-E1 cells were administered a total dose of 0, 2, 4, 6, 8 Gy (2.22 Gy/min) X ray radiation. The cell proliferation was detected by CCK-8 assay and clonogenic assay, the cell cycle and apoptosis were detected by flow cytometry. The osteoblasts differentiation was estimated by ALP staining and the mineralization capacity was evaluated by alizarin red staining. The related gene expression levels were confirmed by RT-qPCR and western blot assay. The DNA damage and repair foci were detected by the immunofluorescence of γ-H2AX and 53BP1. Results In this study, the optimal IR damage conditions (8Gy, 2.22Gy/min) were firstly determined by measuring cell proliferation, cell cycle, cell apoptosis and further cell differentiation and mineralization abilities and the related genes ( p-AKT, p-ERK1/2, cyclinB, BAX, BCL2, ALP, OPN, RUNX2, Collagen1) expression level changes in radiation-induced osteoblast injury model. Then, we screened 26 differentially expressed genes after the RNA-sequencing of the 8 Gy-irradiated MC3T3-E1 cells, and they were mainly involved in DNA damage and repair, cell apoptotic progress and cell cycle regulation, meanwhile, participated in several main pathway including PI3K-AKT signaling pathway, p53 signaling pathway and signaling pathway involved in cell cycle and cell senescence. We focused on verifying the differential expression genes and confirmed the MDM2, NOTCH1, CDKN1A and GCLC were upregulated after IR treatment, suggesting the key roles in the response of the IR. In addition, regarding on our sequencing results, the DNA damage and repair were also verified and the results suggested that IR induced DNA damage and repair in MC3T3-E1 cells. Conclusions In summary, IR damaged MC3T3-E1 cells by inhibiting cell proliferation, impacting cell cycle process, inducing cell apoptosis and affecting the osteoblasts differentiation, which maybe due to the DNA damage and the differential expression of the key genes (MDM2, NOTCH1, CDKN1A and GCLC ) .