Metastasis remains a major obstacle limiting the survival of patients with osteosarcoma (OS). Cross-talk between cancer-associated fibroblasts (CAFs) and OS cells has been found to facilitate metastasis, although the effects of CAFs on OS cell metabolism are poorly understood. Here, conditioned medium from OS cells was used to activate CAF-like cells, which was found to promote OS migration and the epithelial-mesenchymal transition. Metabolomics analysis showed that treatment of OS cells with CAFs-conditioned medium significantly altered the levels of phosphoserine aminotransferase 1 (PSAT1), a key serine synthase. CAF-induced OS cell migration was inhibited by PSAT1 knockdown by siRNA, and PSAT1 was found to promote migration through the PI3K/mTOR/S6K pathway. The influence of CAFs on OS metastasis was blocked by PSAT1 knockdown and mTOR inhibitors and . In conclusion, the findings suggest that PSAT1 and the mTOR/S6K pathway have the potential as targets for preventing OS metastasis.