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Background Osteosarcoma (OS) is one of the most commonly diagnosed malignant tumors that mainly affects children and adolescents. The underlying molecular mechanisms that are responsible for the initiation and development of OS are still not clear. Increasing evidence suggested the tumor suppressor role of microRNA-524-5p in a variety of cancers via targeting key pathways involved in tumorigenesis. The aim of this study was to characterize the function of miR-524-5p in OS. Methods A total 50 paired OS tissues and adjacent normal tissues were collected from OS patients. The expression of miR-524-5p in OS tissues and cells was detected by RT-qPCR. The CCK-8 assay, flow cytometry and transwell assay were applied to determine the proliferation and invasion abilities of OS cells. The targets of miR-524-5p were predicted using the miRDB dataset and confirmed by luciferase reporter assay and western blot analysis. Results The expression of miR-524-5p was decreased in OS tissues and cell lines. OS patients with lymph node metastasis harbored relative lower level of miR-524-5p. Overexpression of miR-524-5p in OS cells significantly suppressed the proliferation, drove cell cycle arrest and apoptosis. The mechanism investigation revealed that miR-524-5p bound the 3′-untranslated region (UTR) of Cyclin Dependent Kinase 6 (CDK6) and repressed the expression of CDK6 in OS cells. Overexpressed CDK6 was found in OS tissues, which was inversely correlated with that of miR-524-5p. Moreover, forced expression of CDK6 significantly reversed the anti-cancer effects of miR-524-5p on the proliferation, apoptosis and cell cycle arrest of OS cells. Conclusions Our results identified the tumor-suppressive role of miR-524-5p in OS via targeting CDK6, which may lead to the identification of novel therapeutic target for the treatment of OS.