Livin is a novel member of the inhibitor of apoptosis protein (IAP) family that has been reported to be overexpressed in a variety of human malignancies, including osteosarcoma. However, the potential roles of Livin in tumorigenesis have not been elucidated. In the present study, we employed RNA interference (RNAi) technology to suppress endogenous Livin expression in osteosarcoma cells and successfully generated a U2-OS cell line with stably knockdown of Livin. Functional analysis showed that knockdown of Livin significantly reduced cell proliferation, colony formation, and invasion and migration capacities of U2-OS cells in vitro. Moreover, specific downregulation of Livin led to cell cycle arrest at the G0/G1 phase and eventual apoptosis. Meanwhile, western blot analysis revealed that cells with stably knockdown of Livin showed decreased expression levels of Cyclin D1, Bcl-2, matrix metalloproteinase (MMP)-2 and MMP-9, but increased expression levels of activated Caspase-3, Bax and cleaved poly (ADP-ribose) polymerase (PARP) compared to those transfected with a control vector. We also observed that suppression of Livin expression in osteosarcoma cells increased their chemosensitivity to cisplatin. Taken together, our data suggest that Livin is involved in tumorigenesis of human osteosarcoma and may serve as a promising therapeutic target for osteosarcoma.