Osteosarcoma (OS) is the mostly diagnosed primary bone malignancy. Emerging evidence indicates that the activity of pyruvate kinase M2 (PKM2) isoform is crucial for the survival of tumor cells. In the present study, the effect of PKM2 knockdown on the proliferation and migration of OS cells were assessed both in vitro and in vivo. Small hairpin RNA (shRNA) technology were employed to suppress the expression of PKM2 in MG-63 and Saos-2 cell lines. In vitro, shRNA-mediated knockdown of PKM2 efficiently inhibited cell proliferation, and induced G1 cell cycle arrest and apoptosis in both cell lines, which was associated with decreased expressions of cyclin D1 and Bcl-2 as well as increased expressions of Bax, cleaved-caspase-3, and cleaved-PARP. The invasion and migration potential of OS cell lines were also inhibited by PKM2 knockdown through the regulating effect of PKM2 on MMP-2 and VEGF signaling. In vivo, knockdown of PKM2 decelerated tumor growth rate and induced structure deterioration in tumor tissues. The current study for the first time showed that the activity of PKM2 was indispensable for the development and metastasis of OS, thereby providing the basic information for the future development of PKM2-based anti-OS therapies.