MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression post-transcriptionally, often playing critical roles in various biological processes. Recent studies have highlighted the involvement of miRNAs in chondrogenesis by targeting key marker genes. Among these, miR-101a has been identified as a significant regulator, previously reported to target cyclooxygenase-2 (Cox-2, ptgs2) in various contexts. Here, we investigate the role of miR-101a in chondrocyte hypertrophy and osteoarthritis (OA) progression, focusing on its regulation of Col10a1 expression. Using multiple web-based tools (TargetScan, PicTar, miRDB, and miRCODE), we identified miR-101a as a potential regulator of Col10a1. Our in vitro experiments demonstrated that miR-101a was down-regulated during chondrocyte hypertrophy in MCT and ATDC5 cells, while Col10a1 and Cox-2 expression levels were up-regulated. Overexpression of miR-101a via mimics resulted in a significant decrease in Col10a1 and Cox-2 at both mRNA and protein levels, whereas inhibition of miR-101a led to their up-regulation. Additionally, MMP-13 protein levels were reduced upon miR-101a overexpression, with no significant changes in Sox9 and Runx2 expression. Luciferase reporter assays confirmed that Cox-2 was a direct target of miR-101a, suggesting that miR-101a indirectly regulates Col10a1 expression via Cox-2. In vivo, intra-articular injection of miR-101a mimics in a medial meniscus-induced OA mouse model resulted in decreased Col10a1 expression and reduced articular damage, supporting the protective role of miR-101a in OA progression. Our findings highlight miR-101a as a negative regulator of chondrocyte hypertrophy through Cox-2, and could be a potential target for further exploration in OA therapy.