MicroRNAs are widely involved in cancer progression by inhibiting the expression levels of oncogenes or tumor suppressor genes, and dysregulation of microRNAs may contribute to tumorigenesis. Here, we found that overexpressed miR-208b can reduce the proliferation of human osteosarcoma cell lines U-2OS and Saos-2 by arresting cell cycle progression. The in vivo xenograft tumors induced by Saos-2 cells overexpressing miR-208b had smaller size and grew more slowly than those induced by the control cells. The mobility of U-2OS or Saos-2 cells was also downregulated by miR-208b. MiR-208b targeted a site in the 3′ untranslated region of receptor tyrosine kinase–like orphan receptor 2. Inhibition of receptor tyrosine kinase–like orphan receptor 2 suppresses osteosarcoma metastasis in vitro. Recovering the expression levels of receptor tyrosine kinase–like orphan receptor 2 in miR-208b-overexpressed U-2OS or Saos-2 cells attenuated the inhibitory effects of miR-208b. In addition, the expression levels of miR-208b are significantly reduced in human osteosarcoma tissue samples compared to normal tissue samples, and miR-208b levels correlated inversely with receptor tyrosine kinase–like orphan receptor 2 levels. On these bases, we identified that miR-208b targets receptor tyrosine kinase–like orphan receptor 2 gene by which miR-208b can regulate the development of osteosarcoma.