Osteosarcoma is the most common primary malignant bone tumor that mostly affects young people’s health. The prognosis of patients with unresectable or recurrent osteosarcoma still remains dismal. Based on gene integration analysis from GEO and TARGET databases by R language, the differentially expressed genes of osteosarcoma patients were identified. Biological molecular function analysis indicated that these genes were importantly enriched in the process of cell adhesion molecule binding. Gene significance highly-related to clinical traits of osteosarcoma was found by weighted gene co-expression network analysis. Additionally, receiver operating characteristic curve analysis was conducted to find prognostic markers in LASSO Cox regression model. Two candidate biomarkers, ANXA1 and PSAT1, for the prognosis of osteosarcoma were detected separately on the basis of WGCNA and LASSO model. Of note, their expression profiles were interrelated with an important therapeutic target HSPA5. In vitro pharmaceutical experiments were performed to explore the biological role and prognostic benefit of candidates. Suppression of HSPA5 effectively upregulated ANXA1 and inhibited PSAT1, resulting in osteosarcoma cell proliferation arrest and apoptosis. These findings suggest that HSPA5 serves as a core molecule for osteosarcoma therapy due to its bidirectional regulation of candidate prognostic biomarkers ANXA1 and PSAT1.