Glucose transporter protein 1-targeted RNA interference inhibits growth and invasion of the osteosarcoma cell line MG63 in vitro. Malignant cells show increased glucose uptake, which is thought to be mediated by glucose transporters. Glucose transporter protein 1 (Glut-1) is critical for growth, proliferation, and migration of tumor cells and Glut-1 overexpression is associated with poor overall survival in osteosarcoma patients. The present study was designed to determine the role of Glut-1 in the growth and invasion of the osteosarcoma cell line MG63, using RNA interference technology in vitro. shRNA-expressing lentiviral vectors targeting the Glut-1 gene were constructed, which were stably expressed in MG63 cells. The level of Glut-1 mRNA was investigated using real-time reverse transcription-polymerase chain reaction, and the protein expression of Glut-1 mRNA was observed using western blotting. MG63 cellular glucose uptake, proliferation, and migration were detected by methyl thiazole tetrazolium assay and flow cytometry. A Glut-1-specific shRNA-expressing lentiviral vector was obtained, which could efficiently inhibit the mRNA and protein expression of Glut-1 to ∼82%-85% in MG63 cells. Downregulation of Glut-1 inhibited the cellular glucose uptake, growth, and invasion of MG63 cells in vitro. These results indicate that RNA interference targeting of Glut-1 could be an effective strategy for the treatment of osteoscarcoma patients.