Leptin signaling influences osteoblastogenesis and modulates the fate of mesenchymal stem cells (MSCs) during bone and cartilage regeneration. Although MSC5 abound in the osteosarcoma (OS) microenvironment, and leptin exhibits pro-tumorigenic properties, leptin's influence on OS progression and chemoresistant signaling in MSC5 remains unclear. Using cell viability and apoptosis assays, we showed that medium conditioned by leptintreated human MSC5 promotes cisplatin resistance in cultured human OS cells. Moreover, GFP-LC3 expression and chloroquine treatment experiments showed that this effect is mediated by stimulation of autophagy in OS cells. TGF-beta expression in MSC5 was upregulated by leptin and suppressed by leptin receptor knockdown. Silencing TGF-beta in MSC5 also abolished OS cell chemoresistance induced by leptin-conditioned medium. Cisplatin resistance was also induced when leptin-conditioned MSC5 were co-injected with MG-63 OS cells to generate subcutaneous xenografts in nude mice. Finally, we observed a significant correlation between autophagy-associated gene expression in OS clinical samples and patient prognosis. We conclude that leptin upregulates TGF-beta in MSC5, which promotes autophagy-mediated chemoresistance in OS cells.