BACKGROUND: 3-hydroxy-3-methylglutaryl-coenzymOHBe A(HMG-CoA) lyase (HMGCL) catalyzes the cleavage of HMG-CoA into acetyl-CoA and acetoacetic acid and serves as a rate-limiting enzyme in the metabolism of ketone bodies. While HMGCL is involved in various biological processes, its specific role in osteosarcoma remains unclear. METHODS: Using data from a public database of osteosarcoma patients, we investigated the expression and prognostic value of HMGCL. The effects of HMGCL on the proliferation, migration, and invasion of osteosarcoma cells were assessed using CCK-8 assays, wound healing tests, and transwell invasion assays. We explored and validated the specific molecular mechanisms by which HMGCL influences osteosarcoma through transcriptome sequencing. Finally, we established a subcutaneous tumor formation model in nude mice to investigate the function of HMGCL in vivo. RESULTS: The expression of HMGCL is downregulated in osteosarcoma and correlates with the prognosis of osteosarcoma patients. Overexpression of HMGCL can inhibit the proliferation, migration, and invasion of osteosarcoma cells, as well as tumor growth in vivo. Through our investigation of the underlying mechanism, we found that HMGCL may inhibit the activation of the PI3K/AKT/mTOR signaling pathway via its product, β-HB. This inhibition promotes the phosphorylation of ULK1, thereby facilitating autophagy in osteosarcoma cells and enhancing the malignancy of the disease. CONCLUSION: HMGCL inhibits the activation of the PI3K/AKT/mTOR signaling pathway mediated by β-HB, thereby reducing the proliferation, migration, and invasion of osteosarcoma cells while promoting autophagy. HMGCL may represent a new target for the treatment of osteosarcoma, offering new hope for patients with this disease.