Abstract: Backgrounds and Objective: Methotrexate (MTX) is an anti-metabolite and anti-rheumatic drug that is widely used in cancer treatment. It is known that a high dose of MTX chemotherapy causes bone growth disturbance in developing bones. Hypericum perforatum (HP) plant is useful against some diseases for local people and regulates apoptosis which is also known as programmed cell death and recent studies also reported that HP reduces the side effects of many toxic drugs. For this reason, we planned a study to reveal the osteoprotective effect of HP extract against MTX induced osteo-toxicity. Materials and Methods: For this purpose MLO-Y4 cells were cultured, then these cells were treated with HP and methotrexate and mediators of the apoptotic, MAPK pathway and the oxidative stress parameters were analyzed on these cells. Results: Expression of proapoptotic factors increased and expression of antiapoptotic factors were decreased with MTX treatment. Treatment of HP ameliorated the apoptotic effect of MTX on MLO-Y4 cells. HP treatment also inhibited MAPK activity and oxidative stress induced by methotrexate. Conclusion: HP attenuated methotrexate-induced osteo-toxicity via reducing oxidative stress and MAPK activity and HP is an effective reducing side effect of MTX.