Osteoblasts are sensitive to ionizing radiation. The small GTPase RhoA and its effector Rho‑associated protein kinase (ROCK) are critical to several cellular functions, including cytoskeleton reorganization, cell survival, and cell differentiation. However, whether the RhoA/ROCK signaling pathway is involved in the regulation of osteoblast cytoskeleton reorganization and differentiation induced by low‑dose X‑ray irradiation remains to be determined. The aim of the present study was to investigate the role of the RhoA/ROCK signaling pathway in mediating differentiation of osteoblasts and reorganization of the cytoskeleton under low‑dose X‑ray irradiation. Osteoblasts were pretreated with the ROCK kinase‑specific inhibitor (Y‑27632) before exposure to low‑dose X‑ray irradiation. The changes of F‑actin in MC3T3 cells were observed at different time points following X‑ray irradiation. Cell Counting Kit‑8 assay, alkaline phosphatase activity, Alizarin red staining and western blotting were used to detect the proliferation and differentiation of osteoblasts after 0.5‑Gy X‑ray irradiation. In the present study, low‑dose X‑ray irradiation promoted the expression of genes associated with the cytoskeleton reorganization. Indeed, the results showed that, 0.5‑Gy X‑ray irradiation can induce reorganization of cytoskeleton and promote differentiation of osteoblasts through the RhoA/ROCK signaling pathway. Additionally, inhibiting ROCK activity blocked low‑dose X‑ray irradiation‑induced LIMK2 phosphorylation, stress fiber formation and cell differentiation. Thus, these results demonstrated the excitatory effects of low‑dose X‑ray irradiation on MC3T3‑E1 cells, including reorganization of the cytoskeleton and differentiation of osteoblasts.