BackgroundAccumulating evidence has suggested that glycometabolism plays an important role in the pathogenesis of tumorigenesis. However, few studies have investigated the prognostic values of glycometabolic genes in patients with osteosarcoma (OS). This study aimed to recognize and establish a glycometabolic gene signature to forecast the prognosis, and provide therapeutic options for patients with OS.MethodsUnivariate and multivariate Cox regression, LASSO Cox regression, overall survival analysis, receiver operating characteristic curve, and nomogram were adopted to develop the glycometabolic gene signature, and further evaluate the prognostic values of this signature. Functional analyses including Gene Ontology (GO), kyoto encyclopedia of genes and genomes analyses (KEGG), gene set enrichment analysis, single-sample gene set enrichment analysis (ssGSEA), and competing endogenous RNA (ceRNA) network, were used to explore the molecular mechanisms of OS and the correlation between immune infiltration and gene signature. Moreover, these prognostic genes were further validated by immunohistochemical staining.ResultsA total of four genes includingPRKACB,SEPHS2,GPX7, andPFKFB3were identified for constructing a glycometabolic gene signature which had a favorable performance in predicting the prognosis of patients with OS. Univariate and multivariate Cox regression analyses revealed that the risk score was an independent prognostic factor. Functional analyses indicated that multiple immune associated biological processes and pathways were enriched in the low-risk group, while 26 immunocytes were down-regulated in the high-risk group. The patients in high-risk group showed elevated sensitivity to doxorubicin. Furthermore, these prognostic genes could directly or indirectly interact with other 50 genes. A ceRNA regulatory network based on these prognostic genes was also constructed. The results of immunohistochemical staining showed thatSEPHS2,GPX7, andPFKFB3were differentially expressed between OS tissues and adjacent normal tissues.ConclusionThe preset study constructed and validated a novel glycometabolic gene signature which could predict the prognosis of patients with OS, identify the degree of immune infiltration in tumor microenvironment, and provide guidance for the selection of chemotherapeutic drugs. These findings may shed new light on the investigation of molecular mechanisms and comprehensive treatments for OS.