The limited effectiveness and high toxicity of current treatments in osteosarcoma necessitate new therapeutic strategy. Cobimetinib is a FDA-approved MEK inhibitor and is clinically used in combination with standard of care to treat melanomas. Here, we report that targeted MEK inhibition by cobimetinib enhances doxorubicin's efficacy in osteosarcoma models. We found that cobimetinib potently inhibited growth and survival of osteosarcoma cells. We revealed that cobimetinib had anti-metastasis activity as it inhibited osteosarcoma cell migration. Notably, the effective concentrations of cobimetinib are clinically achievable. We further found that cells with the most sensitivity had highest p-ERK and cells with the least sensitivity had lowest p-ERK, suggesting the possible correlation of ERK activation with cobimetinib sensitivity in osteosarcoma. We further confirmed that inhibition of MEK/ERK signaling pathway is the mechanism of cobimetinib's action in osteosarcoma, leading to inhibition of focal adhesion kinase (FAK) and anti-apoptotic pathway, as well as activation of pro-apoptotic pathway. Using xenograft mice model, we found that cobimetinib at the tolerable dose significantly inhibited osteosarcoma formation and growth. In addition, the combination of cobimetinib and doxorubicin at sublethal dose completely arrested tumor growth without further progression. The ability of cobimetinib in enhancing doxorubicin's efficacy in osteosarcoma models makes cobimetinib as a useful addition to the treatment armamentarium for osteosarcoma. Our findings also emphasize the therapeutic value of MEK/ERK pathway to improve the clinical management of osteosarcoma. (C) 2020 Elsevier Inc. All rights reserved.