Osteosarcoma is the most common human primary malignant bone tumor and recurrences are common due to the development of chemoresistance. However, the underlying molecular mechanism for chemoresistance remains unclear. Recent studies demonstrated that miR-146b-5p, an important regulator in tumorigenesis, was involved in chemoresistance in thyroid cancer, lymphoma. Thus, to confirm the role of miR‑146b-5p in osteosarcoma, the study was divided into three steps: first, miR-146b-5p in paired samples were assessed using a quantitative real-time PCR (qRT-PCR) assay from osteosarcoma patients. Second, to confirm the role of miR-146b-5p, we applied lentivirus system to overexpression and knockdown of miR-146b-5p, respectively, in MG-63 osteosarcoma cell line. Third, luciferase assays were performed to determine whether Wnt/β-catenin pathway participated in the role of miR-146b-5p on chemoresistance. As a result, miR-146b-5p was highly expressed in human osteosarcoma tissues and an elevated expression of miR-146b-5p was observed in human osteosarcoma tissues after chemotherapy. Furthermore, it was shown that miR-146b-5p overexpression promoted migration and invasiveness. miR-146b-5p overexpression also increased resistance to chemotherapy. Moreover, knockdown of miR-146b-5p substantially inhibited migration and invasion of osteosarcoma cells as well as rendered them significantly more sensitive to chemotherapy. Results of western blot assay indicated that miR-146b-5p increased MMP-16 protein expression and showed a decrease of ZNRF3 protein. Whereas, IWR-1-endo, an inhibitor of Wnt/β-catenin, suppressed the decrease in apoptosis of osteosarcoma cells caused by miR-146b-5p overexpression. These results indicated that miR-146b-5p promoted proliferation, migration and invasiveness. It also increased resistance to chemotherapy through the regulation of ZNRF3, and suggested novel potential therapeutic targets for the treatment of osteosarcoma.