MicroRNAs (miRs) play crucial roles in tumorigenesis by directly suppressing the protein expression levels of their target genes. miR-204 has been suggested to act as a tumor suppressor in several types of human cancer. However, the exact role of miR-204 in osteosarcoma (OS) remains undetermined. In the present study, we aimed to investigate the effects of miR-204 on OS cell proliferation, migration and invasion, as well as the underlying molecular mechanisms. We found that the expression of miR-204 was frequently downregulated in four OS cell lines compared to the level in normal human osteoblast cells. Moreover, overexpression of miR-204 significantly inhibited the proliferation, migration and invasion of OS cells. Based on bioinformatics prediction and a luciferase reporter assay, we identified Sirtuin 1 (Sirt1) as a direct target gene of miR-204 in OS Saso-2 cells. Moreover, the protein expression of Sirt1 was negatively mediated by miR-204 in the OS cells. siRNA-mediated knockdown of Sirt1 also inhibited the proliferation, migration and invasion of the OS cells. Moreover, overexpression of Sirt1 reversed the inhibitory effect of miR-204 overexpression on the proliferation, migration and invasion of the OS cells. In addition, after miR-204 overexpression or Sirt1 knockdown in OS cells, the expression of E-cadherin was increased, while the N-cadherin protein level was reduced. Based on these findings, we suggest that miR-204 inhibits the proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) of OS cells by directly targeting Sirt1.