Osteosarcoma has one of the worst prognoses in adolescents; only 20-60% of patients have high rates of histological necrosis with intensive neoadjuvant chemotherapy. In this study, we investigated the prognostic values of hypoxia-inducible factor 1 α (HIF-1α), apurinic endonuclease 1 (APE1), vascular endothelial growth factor (VEGF) and cycloogenase-2 (COX-2) protein expression and their predictive value of tumor necrosis rate and prognosis, as well as their interrelationships. Formalin-fixed paraffin-embedded tissue samples were obtained from 49 patients with osteosarcoma. Immunohistochemistry assays were performed in pre-chemotherapy samples to determine HIF-1α, VEGF, APE1 and COX-2 protein expression levels and hematoxylin and eosin staining was performed in post-operative samples to determine the tumor necrosis rate. Univariate and multivariate analyses were used to assess the impact of protein expression on prognosis. HIF-1α was significantly correlated with every protein we tested: VEGF (P=0.032), APE1 (P<0.001) and COX-2 (P<0.001). HIF-1α protein expression had a significant impact on disease-free survival (P=0.006). Expression of HIF-1α had a sensitivity of 64.7% and a specificity of 71.9% for a poor pathological response (<90% tumor necrosis) versus a good pathological response (≥90% tumor necrosis). In conclusion, expression of HIF-1α is a predictor of tumor response to neoadjuvant chemotherapy and outcome in osteosarcoma, and correlates with VEGF, APE1 and COX-2.