Osteosarcoma, a leading malignant tumor of bones is diagnosed mostly in adolescents and young adults worldwide. The present study investigated alstonine as antiosteosarcoma agent in vitro as well as in vivo and evaluated the underlying mechanism. Treatment with alstonine led to a significant (P<0.05) reduction in MG63 and U-2OS cell viability. Alstonine treatment of MG63 and U-2OS cells caused a significant reduction in colony formation compared to the control cells. Viability of osteoblasts was not affected by alstonine treatment in 1.25 to 20 μM concentration range. In alstonine treated MG63 and U-2OS cells apoptotic cells increased significantly (P<0.05) compared to the control cells. Moreover, in MG63 and U-2OS cells treatment with alstonine caused a prominent increase in expression of cleaved caspase-9, caspase-3, and PARP. Treatment of MG63 and U-2OS cells with alstonine caused a prominent increase in AMPKα (Thr172) phosphorylation and elevated the count of mtDNA copies compared to the untreated cells. Alstonine treatment of the cells caused a remarkable increase in expression of PGC-1α and TFAM proteins. Treatment of the mice with alstonine at 5 and 10 mg/kg doses for 30 days caused a significant (P<0.05) reduction in xenograft growth. Expression of PGC-1α and TFAM proteins in tumor tissues of the mice treated with alstonine was significantly (P<0.05) raised compared to the control group. Thus, alstonine inhibits osteosarcoma cell growth and activates apoptosis through AMPK dependent pathway. Therefore, alstonine may be considered for treatment of osteosarcoma as it effectively arrests tumor growth in mice. © 2022,Acta Biochimica Polonica. All Rights Reserved.