AbstractChondrosarcoma is the most common primary bone malignancy in the adult population with a high rate of pulmonary metastasis. Chondrosarcoma is managed with surgical excision as the tumors do not respond well to conventional chemotherapy or radiation therapy. Thus, there exists a dire need to develop systemic treatment options to target chondrosarcoma cells for metastatic spread. We hypothesized that the expression of miR‐145 is low in chondrosarcoma, leading to decreased transcriptional control of SOX9 (the master regulator of chondrogenesis), and downstream activation of the transcription factor ETV5. We have previously shown that ETV5 activates MMP‐2 expression in chondrosarcoma, which in turn increases local bone matrix resorption. In this study, we confirm high expression of SOX9 in human chondrosarcoma using real‐time PCR, Western blotting, and immunofluorescence. An ETV5 promoter–reporter plasmid was transfected into chondrosarcoma cells to determine if SOX9 directly regulates the expression of ETV5. Co‐transfection of the ETV5 promoter‐plasmid with SOX9 lentivirus significantly increased the luciferase activity derived from the ETV5 promoter, from which the regulatory relationship between SOX9 and ETV5 is established. MiR‐145 was found to be down‐regulated in chondrosarcoma cell lines, patient samples, and further confirmed with a public sarcoma database. After stable miR‐145 lentiviral transfection, the subsequent mRNA expression levels of SOX9, ETV5, and MMP‐2 were significantly decreased in chondrosarcoma cells. The results generated by this study may have important clinical significance in the treatment of patients with chondrosarcoma in that targeted miRNA may have the potential to downregulate the upstream activators of proteases such as MMP‐2. J. Cell. Biochem. 116: 37–44, 2015. © 2014 Wiley Periodicals, Inc.