Background <p>This study aims to unravel the underlying mechanisms of osteosarcoma (OS) metastasis by single-cell RNA-sequencing.</p> Methods <p>Ten pediatrics OS samples were analyzed using scRNA-seq and divided into three groups: primary tumor without lung metastasis (Pri_non_MT), primary tumor with lung metastasis (Pri_MT), and lung metastasis site (MT). Candidate genes associated with metastasis were identified by bioinformatics analysis and was confirmed at mRNA and protein levels in OS tissues. The function of candidate gene was identified in vitro and in vivo. Bulk RNA sequencing was used to explore downstream mechanism after candidate gene silenced.</p> Results <p>A total of 17 cell clusters were identified. ENO1 was selected as the candidate gene and was significantly expressed in Pri_MT and MT groups. In OS tissues, ENO1 significantly overexpressed in patients with lung metastasis compared to those without. Knocking down ENO1 resulted in a marked decrease in migration and invasion in vitro and a reduction in lung metastasis in vivo. Additionally, ENO1 suppression resulted in shifting the primary ATP production pathway from glycolysis to oxidative phosphorylation.</p> Conclusions <p>Our findings highlight ENO1 as a key regulator of glycolysis and metastasis in osteosarcoma, offering a novel therapeutic target for OS treatment.</p>