Heat shock protein 70 (HSP70) abundantly expressed in human tumors and protects cells from a wide range of apoptotic stimuli. To study the role of HSP70 in human hepatocarcinomas, we stably silenced HSP70 gene expression in HepG(2) cells by transfecting the cells with HSP70-silencing vector (HSP70 shRNA). HSP70 silencing resulted in reduced capacities for cell proliferation in vitro and colony formation in soft agar. In addition, HSP70 silencing enhanced chemotherapy drug-induced cell death and tumor growth in a mouse xenograft model. Most importantly, we extended our previous observation that HSP70 modulates mitochondrial protein Apoptosis-inducing factor (AIF)-induced cell death. In this study, we found that HSP70 silencing dramatically increased AIFDelta1-120 mutant (an active form of AIF after deletion of the mitochondria localization domain)-induced apoptosis. In HSP70 silenced cells, AIFDelta1-120 mutant predominantly localized in nuclear compartment compared to the control cells, confirming that HSP70 protects from apoptosis by sequestering AIF in the cytoplasm. Taken together, our data suggest that HSP70 ablation in combination with delivery of AIFDelta1-120 mutant or other chemotherapy drugs represents a potent anti-cancer therapy for hepatocarcinomas.