Sorafenib has been used to treat advanced hepatocellular carcinoma (HCC), but the underlying molecular mechanisms remain controversial and why some patients do not respond to this therapy is poorly understood. In this study, we show that sorafenib triggers cell growth inhibition and apoptosis in HCC cells by directly targeting the mitochondria. Treatment with sorafenib induces rapid mitochondrial fragmentation, which is associated with the deregulation of mitochondria fusion-related protein optic atrophy 1 (OPA1). Exposure of cells or isolated mitochondria to sorafenib substantially induces cytochrome c release. Our data indicate that siRNA-mediated OPA1 knockdown significantly sensitizes HCC cells to sorafenib-induced apoptosis. Furthermore, sorafenib has no apparent apoptotic toxicity to normal human primary hepatocytes. Sorafenib inhibits HCC xenograft tumor growth in vivo and murine xenograft tumor tissue analysis reveals mitochondria fusion protein. OPA1 expression levels are strongly downregulated by sorafenib treatment. Western blotting evaluation of patient HCC with matched non-tumor tissue samples demonstrates that OPA1 expression is decreased in up to 40% of HCC patients. Taken together, we have shown that sorafenib suppresses the tumorigenesis of HCC through the induction of mitochondrial injury via OPA1. Our results provide new insights into the pathogenesis of HCC and suggest that OPA1 is a novel therapeutic target in patients with HCC.