Relaxin-2 (RLN2) and calcium-binding protein S100A4 was overexpressed in many cancers. Experimental evidence indicated enhanced tumor cell invasion by RLN2 involves the upregulation of S100A4. However, the relationship between them in cancers is not clear. In the present study, we investigate the expression of relaxin-2 protein and calcium-binding protein S100A4 in osteosarcoma by immunohistochemistry assay and their relationship to the clinicopathological parameters and prognosis of osteosarcoma (OS).Expression status of RLN2 and S100A4 was examined in 130 surgical specimens of primary OS by immunohistochemistry. Correlation between the expression of RLN2 and S100A4 and clinicopathological parameters was analyzed.78 of 130 (60%) specimens of primary OS were positive for RLN2. 67 of 130 (51.5%) specimens showed positive expression of S100A4. The positive correlation between RLN2 and S100A4 expression was significant in cancer tissues (p = 0.02). RLN2 and S100A4 expression in osteosarcoma tissues was significantly higher than that in corresponding noncancerous bone tissues both p = 0.000). In addition, high RLN2 and S100A4 expression more frequently occurred in osteosarcoma tissues with advanced clinical stage (both p < 0.05) and positive distant metastasis (both p < 0.05). Moreover, osteosarcoma patients with high RLN2 and S100A4 expression had significantly shorter overall survival and disease-free survival (both p < 0.001) when compared with patients with the low expression of RLN2 and S100A4. On Cox multivariate analysis, RLN2 and S100A4 overexpression, distant metastasis were the independent and significant prognostic factor to predict poor overall survival and disease-free survival.We, therefore, suggested overexpression of RLN2 and S100A4 might be related to the prediction of metastasis potency and poor prognosis for osteosarcoma patients. Detection of RLN2 and S100A4 might be useful in evaluating the outcome of patients with OS.