Osteosarcoma (OS) is the most common malignant primary bone tumor disease. HIF-1α was predicted to be the target gene of microRNA-20b (miR-20b). The present study was designed to illustrate the effect of miR-20b in regulating osteosarcoma via targeting HIF-1α. In this study, we found that the expression of HIF-1α was significantly increased, while miR-20b obviously decreased in OS patients and OS cell lines compared with healthy controls. Moreover, the luciferase report confirmed the targeting reaction between miR-20b and HIF-1α. Additionally, the overexpression of miR-20b suppressed the invasion and growth of both MG63 and U2OS cells, and inhibited the expressions of HIF-1α and VEGF pathway proteins, while the inhibition of miR-20b led to the reverse results. Furthermore, the overexpression of HIF-1α affected the suppression effect of miR-20b in MG63 cells, indicating that miR-20b suppresses the tumor cell process via inhibiting the expression of HIF-1α. Taken together, our results suggest that the upregulation of miR-20b affects the expression of HIF-1α, downregulates the VEGF pathway proteins, and suppresses cell invasion and proliferation rate. These results provide a potential therapeutic strategy for osteosarcoma.