Recently, research into the biological effects of low dose X-ray irradiation (LDI) has been a focus of interest. Numerous studies have suggested that cells exhibit different responses and biological effects to LDI compared with high doses. Preliminary studies have demonstrated that LDI may promote osteoblast proliferation and differentiation in vitro, thereby accelerating fracture healing in mice. However, the exact mechanism of action by which LDI exerts its effects remains unclear. Previous studies using microarrays revealed that LDI promoted the expression of genes associated with the cytoskeleton. In the current study, the effect of X-ray irradiation (0.5 and 5 Gy) on the morphology of MC3T3-E1 cells and fiber actin organization was investigated. Osteoblasts were treated with 0, 0.5 and 5 Gy X- ray irradiation, following which changes in the actin cytoskeleton were observed. The levels of RhoA, ROCK, cofilin and phosphorylated-cofilin were measured by reverse transcription-quantitative PCR and western blotting. Subsequently, osteoblasts were pretreated with ROCK specific inhibitor Y27632 to observe the changes of actin skeleton after X-ray irradiation. The results demonstrated that the cellular morphological changes were closely associated with radiation dose and exposure time. Furthermore, the gene expression levels of small GTPase RhoA and its effectors were increased following LDI. These results indicated that the RhoA/Rho-associated kinase pathway may serve a significant role in regulating LDI-induced osteoblast cytoskeleton reorganization.