Neutral endopeptidase (NEP/CD10) and dipeptidyl peptidase IV (DPP IV/CD26) are both ubiquitous glycopeptidases which play important roles in tumor pathogenesis and development. The aim of this study was to investigate the expression patterns and the prognostic significance of CD10 and CD26 in osteosarcoma patients. CD10 and CD26 expression in 116 pairs of primary osteosarcoma and corresponding noncancerous bone tissue samples from the same specimens were detected by immunohistochemistry. The Spearman's correlation was calculated between the expression levels of CD10 and CD26 in osteosarcoma tissues. The associations of CD10 and CD26 expression with the clinicopathologic features and with the prognosis of osteosarcoma were subsequently assessed. Both CD10 expression and CD26 expression in osteosarcoma tissues were significantly higher than those in corresponding noncancerous bone tissue samples (both P < 0.001). Overexpression of CD10 and CD26 were respectively observed in 68.10 % (79/116) and 70.69 % (82/116) of osteosarcoma tissues. A significant correlation was found between CD10 expression and CD26 expression in osteosarcoma tissues (r = 0.83, P < 0.001). In addition, combined overexpression of CD10 and CD26 was observed in 52.59 % (61/116) of osteosarcoma tissues. CD10-high/CD26-high expression was significantly correlated with advanced clinical stage (P = 0.001), positive metastatic status (P = 0.001), shorter overall (P < 0.001) and disease-free (P < 0.001) survival in patients with osteosarcomas. Furthermore, multivariate survival analysis showed that clinical stage, metastatic status, CD10 expression, CD26 expression and combined expression of CD10/CD26 were all independent prognostic factors for predicting both overall and disease-free survival of osteosarcoma patients. Interestingly, combined expression of CD10/CD26 had a better prognostic value than other features. This retrospective study offer the convincing evidence for the first time that the overexpression of CD10 or CD26 may be an important feature of human osteosarcomas, and the combined expression of CD10/CD26 may be an efficient prognostic indicator for this disease.