AbstractMicroRNAs (miRNAs) are small non‐coding RNAs capable of inhibiting gene expression post‐transcriptionally and expression profiling can provide therapeutic targets and tools for cancer diagnosis. Chondrosarcoma is a mesenchymal tumor with unknown cause and differentiation status. Here, we profiled miRNA expression of chondrosarcoma, namely clinical samples from human conventional chondrosarcoma tissue, established chondrosarcoma cell lines, and primary non‐tumorous adult articular chondrocytes, by miRNA array and quantitative real‐time PCR. A wide variety of miRNAs were differently downregulated in chondrosarcoma compared to non‐tumorous articular chondrocytes; 27 miRNAs: miR‐10b, 23b, 24‐1*, 27b, 100, 134, 136, 136*, 138, 181d, 186, 193b, 221*, 222, 335, 337‐5p, 376a, 376a*, 376b, 376c, 377, 454, 495, 497, 505, 574‐3p, and 660, were significantly downregulated in chondrosarcoma and only 2: miR‐96 and 183, were upregulated. We further validated the expression levels of miRNAs by quantitative real‐time PCR for miR‐181a, let‐7a, 100, 222, 136, 376a, and 335 in extended number of chondrosarcoma clinical samples. Among them, all except miR‐181a were found to be significantly downregulated in chondrosarcoma derived samples. The findings provide potential diagnostic value and new molecular understanding of chondrosarcoma. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1992–1998, 2013