BACKGROUND & AIMS: Expression of osteopontin correlates with tumor progression and metastasis. The mechanisms by which osteopontin promotes tumor cell survival remain unclear. Here we used short-hairpin RNA-mediated gene silencing to investigate the antitumor effects by osteopontin depletion in hepatocellular carcinoma (HCC). METHODS: We applied polyethylenimine nanoparticles to deliver a short-hairpin RNA for depletion of osteopontin expression in HCC cells. Tumorigenicity and metastatic potentials of HCC cells were studied in vitro and in nude mice. Nuclear factor-kappaB (NF-kappaB) activation was analyzed by gel shift assay and luciferase analysis. The expressions of integrins were examined by real-time reverse-transcription polymerase chain reaction. Apoptosis was examined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay and mitochondrial membrane potential analysis. RESULTS: Down-regulation of osteopontin inhibited HCC cell growth, anchorage-independent growth, adhesion with fibronectin and invasion through extracellular matrix in vitro, and suppressed tumorigenicity and lung metastasis in nude mice. Osteopontin silencing resulted in suppression of alphav, beta1, and beta3 integrin expressions, blockade of NF-kappaB activation, inhibition of Bcl-2/Bcl-xL and XIAP expressions, increase of Bax expression, and induction of a mitochondria-mediated apoptosis. Furthermore, down-regulation of osteopontin inhibited drug-induced NF-kappaB activation and sensitized HCC cells to chemotherapeutic agents in vitro, which led to complete regression of HCC xenografts in nude mice. CONCLUSIONS: Osteopontin may facilitate tumorigenesis and metastasis through prevention of tumor cells from apoptosis. RNA interference-mediated depletion of osteopontin may be a promising strategy for the treatment of HCC by sensitizing the chemotherapeutic drugs.