ObjectiveThe present study identified potentially pivotal miRNAs contributing to chondrogenic differentiation in temporomandibular joint suffering abnormal stress.Materials and MethodsSprague–Dawley rats were randomly divided into control and experimental unilateral mastication (EUM) group. Bone micro‐structure parameters was detected by micro‐CT, and FGF‐1 and MMP‐1 expression was examined by immunohistochemistry. Differentially expressed miRNAs of bilateral condyle cartilage were screened via miRNA microarray at 4‐ and 8‐week EUM, then further verified using quantitative reverse‐transcription PCR. Over‐expression of five differentially expressed miRNAs in chondrocytes was triggered by transfecting miRNA mimics. The expression of MMP‐13, Col‐II, OPN, and Runx2 was verified by western blotting.ResultsExpressions of FGF‐1 and MMP‐1 in right condyles gradually increased from 2 to 6 weeks after EUM. A total of 20 differentially expressed miRNAs were regulated by EUM, which related to cell proliferation, invasion, and osteoblast differentiation pathways. The over‐expression of miR‐148a‐3p and miR‐1‐3p led to down‐regulation of Col‐II, while MMP‐13 and Runx2 were up‐regulated by induction of hypotrophic differentiation or IL‐1β stimulation. These findings suggested that miR‐148a‐3p and miR‐1‐3p promote chondrogenic differentiation.ConclusionsSeveral pivotal miRNAs were found to be related to chondrogenic differentiation, which provides novel insight into pathogenic mechanisms of cartilage homeostasis.