Osteoarthritis (OA) is an aging-related chronic degenerative disease characterized by the degradation of chondrocyte extracellular matrix (ECM). Previous studies have suggested that microRNAs (miRNAs) are associated with OA, but the role of miR-146b in OA remains unclear. The aim of this study was to determine the role of miR-146b in OA progression. The effect of miR-146b on ECM degradation were studied in mouse chondrocytes transfected with miRNA and treated with IL-1β. Cell viability and the expression levels of proteolytic enzymes in the transfected cells were assessed by real-time RT-PCR, ELISA and Western blots. We found downregulation of miR-146b expression in chondrocytes dramatically inhibited IL-1β-induced caspase activation and proteolytic enzyme expression via influencing its targeted Alpha-2-macroglobulin (A2M). Luciferase reporter assays confirmed that A2M mRNA was negatively regulated by miR-146b in chondrocytes. Intra-articular injection of antago-miR-146b against miR-146b effectively protected mice from the progression of DMM-induced osteoarthritis by inhibiting cartilage proteoglycan degradation. Our study indicates that miR-146b plays a critical role in the progression of injury-induced osteoarthritis by directly targeting A2M expression to elevate the proteolytic enzyme production and stimulate chondrocytes apoptosis, and miR-146b as well as A2M could be therapeutic targets.