Tumorigenesis is a multistep process involving various cell growth-associated factors. Accumulated evidence indicates that the disordered regulation of microRNAs (miRNAs) contributes to tumorigenesis. However, the detailed mechanism underlying the involvement of miRNAs in oncogenesis remains to be fully elucidated. In the present study, the repressed expression of microRNA (miR)-494 was identified in 18 patients with osteosarcoma (OS) and OS cell lines, compared with corresponding controls. To determine whether deregulated miR-494 exerts tumor-suppressive effects in the development of OS, the effects of miR-494 on cell proliferation and metastasis were evaluated. It was found that the restoration of miR-494 in MG-63 and U2OS cells led to inhibited cell proliferation and attenuated migratory propensity in vitro, determined through analysis using MTT, colony formation and Transwell assays. In addition, overexpression of miR-494 markedly suppressed the tumor volume and weight in vivo. In accordance, the ectopic expression of miR-494 induced cell cycle arrest at the G1/S phase in OS cells. Bioinformatics analysis and luciferase reporter assays were performed to investigate the potential regulatory role of miR-494, the results of which indicated that miR-494 directly targeted cyclin-dependent kinase 6 (CDK6). Of note, the data obtained through reverse transcription-quantitative polymerase chain reaction and western blot analyses suggested that the elevated expression of miR-494 resulted in reduced mRNA and protein expression levels of CDK6. Taken together, these findings indicated that the miR-494/CDK6 axis has a significant tumor-suppressive effect on OS, and maybe a diagnostic and therapeutic target for the treatment of OS.