AbstractPrevious study showed that miRNA aberrant expression is involved in chondrogenic differentiation. In this study, we aimed to investigate the effects of miR‐132‐3p on chondrogenic differentiation and the underlying mechanisms. First, quantitative PCR were performed to determine the level of MiR‐132‐3p. Then, we used luciferase assay to examine the target of miR‐132‐3p. Proteoglycan was tested by Alcian blue staining assay. Moreover, the sex determining region Y‐box 9 (SOX9), Collagen type II alpha 1 chain (COL2A1) and Aggrecan (ACAN) levels were analyzed by quantitative PCR, immunofluorescence and Western blotting. Our results showed that MiR‐132‐3p level was reduced in rat MSCs (rMSCs) during chondrogenic differentiation. Ectopic expression of miR‐132‐3p induced proteoglycan accumulation and the increase of ACAN, SOX9 and COL2A1 expression, which were involved in inducing chondrogenic differentiation of rMSCs. More importantly, ADAMTS‐5 was identified as the target of MiR‐132‐3p. Knockdown of ADAMTS‐5 increased proteoglycan level, but reduced the SOX9, ACAN, and COL2A1 levels during chondrogenic differentiation of rMSCs. Taken together, our results revels that MiR‐132‐3p promotes rMSCs chondrogenic differentiation, possibly mediated by targeting ADAMTS‐5, which provided new perspective on the chondrogenic differentiation and pathology of osteoarthritis.