Oral hypoglycemic agent metformin is commonly used for treating type II diabetes; however, initial reports demonstrated that it could be used for suppressing tumor growth in vitro and in vivo. Moreover, novel potential anticancer drug histone deacetylase (HDAC) and inhibitor trichostatin A (TSA) have been extensively studied for inducing various malignancies growth inhibition, cell cycle arrest, and apoptosis. The object of the present study was to investigate the anti-proliferation and apoptosis induction effects of metformin and TSA in osteosarcoma cell line, and to explore the mechanism of metformin and TSA in combination to inhibit the proliferation of osteosarcoma cells. After treating with metformin and TSA, the viability of osteosarcoma cell lines (MG-63 and LM8) was analyzed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) at various concentrations, cell cycle analysis of MG-63 and LM8 cell was performed by flow cytometry. Real-time polymerase chain reaction and Western Blotting were performed to determine the expression of apoptosis-related genes and proteins such as Caspase-3, Bcl-2/Bax, Cyclin D1, and p21. Protein expression of the molecules involved in 5'-adenosine monophosphate-activated protein kinase (AMPK) signaling pathway after treatment with combination was determined by Western blotting. Moreover, orthotopic xenograft tumors were challenged in nude mice to establish the murine model; tumor weight and tumor volume were monitored after drug administration separately or combined via the intraperitoneal (i.p.) route. MTT assays showed that the viability of osteosarcoma cell lines in the combination group (10 mM metformin, 0.3 μM TSA) decreased in a concentration- and time-dependent manner; moreover, the cell cycle of MG-63 and LM8 in the combination group could be arrested in G1/G2 phase higher number compared with drug use separately. Furthermore, a combination of these drugs does not act via the AMPK signaling pathway to induce MG-63 osteosarcoma cell line growth inhibition and apoptosis. As data have showed here, metformin cotreatment increased TSA antitumor effects and have a synergistic effect on osteosarcoma cell line proliferation and apoptosis.