Hepatocellular carcinoma (HCC) is one of the most common malignant cancers worldwide. The prognosis of HCC remains poor. Currently, sorafenib is the first-line drug for advanced HCC. Although sorafenib's mechanism of action involving several established cancer-related protein kinase targets is well-characterized, the underlying molecular mechanism is still unclear. Here, we found that sorafenib inhibited viability, proliferation, and migration of HCC cells in a dose-dependent manner. Sorafenib treatment of HCC cells destroyed mitochondrial morphology, accompanied by decreased activity of oxidative phosphorylation, collapse of mitochondrial membrane potential, and reduced synthesis of ATP, with consequent cell death due to ferroptosis. Pharmacological utilization of glutathione (GSH) rescued the sorafenib-induced ferroptosis, eliminated the accumulation of cellular mitochondrial reactive oxygen species (ROS), and lipid peroxide. GSH depletion through cysteine deprivation or cysteinase inhibition exacerbated sorafenib-induced ferroptotic cell death and lipid peroxides generation, and enhanced oxidative stress and mitochondrial ROS accumulation. Collectively, these findings indicate that depletion of cysteine acts synergistically with sorafenib and renders HCC cells vulnerable to ferroptosis, presenting the potential value of new therapeutic combinations for advanced HCC. (C) 2020 Elsevier Inc. All rights reserved.