Osteosarcoma is the most common type of primary malignant tumor of the bone. However, mechanisms underlying osteosarcoma cell proliferation are poorly understood. The present study shows that RBEL1, a newly identified Rab-like GTPase, may be a key regulator of osteosarcoma cell proliferation. Knockdown of RBEL1 in osteosarcoma cells resulted in impaired colony formation and cell proliferation. Cell cycle analysis suggested that RBEL1 depletion induced G1-S arrest in osteosarcoma cells. Furthermore, it was demonstrated that retinoblastoma 1 (Rb) was upregulated and activated following RBEL1 knockdown. In addition, Rb inhibitory downstream targets, such as cyclin A2, cyclin D1, c-Myc and cyclin-dependent kinase 2, were downregulated. Rb knockdown reversed RBEL1 depletion-induced tumor suppressive effects. In conclusion, the present results suggest that RBEL1 modulates cell proliferation and G1‑S transition by inhibiting Rb in osteosarcoma. These results suggest a potential therapeutic target in osteosarcoma.