Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. Mito-carboxy proxyl (Mito-CP), a lipophilic cationic nitroxide, accumulates in the mitochondria because of the large negative transmembrane potential. Studies have shown that these agents act by disrupting the energy-producing mechanism, inducing mitochondrial-mediated apoptosis, and also enhancing the action of other chemotherapeutic agents in cancer cells. We hypothesized that the combination of Mito-CP and glycolysis inhibitor, 2-deoxyglucose (2-DG), would synergistically inhibit HCC in vitro. HepG2 cells and primary hepatocytes were treated with various combinations of Mito-CP and 2-DG. Cell cytotoxicity was measured using the methylthiazolyldiphenyl-tetrazolium bromide assay and ATP bioluminescence assay. In addition, caspase 3/7 enzymatic activity was examined after treatment. Mito-CP and 2-DG induced synergistic cytotoxicity in HepG2 cells in a dose-dependent and time-dependent manner, whereas primary cells remained viable and unaffected after treatment. The intracellular ATP levels of HepG2 cells were suppressed within 6 h of combination treatment, whereas primary cells maintained higher levels of ATP. Dose-dependent increases in caspase 3/7 activity occurred in HepG2 cells in a time-dependent manner, showing the initiation of cell death through the apoptotic pathway. These findings indicate that a combination of Mito-CP and 2-DG effectively inhibits HCC growth in vitro. The increase in caspase 3/7 activity supports the occurrence of 2-DG-induced and Mito-CP-induced apoptotic death in HCC. The inability of the compounds to induce cytotoxicity or suppress the production of ATP in primary hepatocytes provides a selective and synergistic approach for the treatment of HCC.