Abnormal expression of microRNAs plays important functions in osteosarcoma. The aim of this study was to investigate expression, functions and molecular mechanisms of microRNA-363 in osteosarcoma. Quantitative Real-time PCR was used to detect the expression level of microRNA-363 in osteosarcoma tissue samples and cell lines. After transfection, CCK8 assay, cell migration and invasion assay, western blot and Dual-Luciferase report assay were performed in human osteosarcoma cells. According to the results, we found that microRNA-363 was down-regulated in osteosarcoma tissues and cell lines. In addition, low expression level of microRNA-363 was associated with tumor size, clinical stage and distant metastasis. Moreover, microRNA-363 targeted MAP2K4 to inhibit osteosarcoma cell growth, migration and invasion. In conclusion, microRNA-363 played a tumor suppressive role in osteosarcoma by directly targeting MAP2K4. These findings indicated that microRNA-363 may have therapeutic value in treating osteosarcoma.