Viral fusogenic membrane glycoproteins (FMGs) are candidates for gene therapy of solid tumors because they cause cell fusion, leading to formation of lethal multinucleated syncytia. However, the cellular mechanisms mediating cell death after FMG-induced cell fusion remain unclear. The present study was designed to examine the mechanisms by which FMG expression in hepatocellular carcinoma cells lead to cell death. Transfection of Hep3B cells with the Gibbon Ape leukemia virus hyperfusogenic envelope protein (GALV-FMG) resulted in the formation of multinucleated syncytia that reached a maximum 5 days after transfection (100 nuclei/syncytia). The syncytia were viable for a period of 2 days and then rapidly lost viability by day 5. Mitochondrial dysfunction occurred in GALV-FMG-induced syncytia prior to loss of viability with loss of the mitochondrial membrane potential, cellular ATP depletion, and release of mitochondrial cytochrome c-GFP into the cytosol. The pan-caspase inhibitor, Z-VAD-fmk, did not prevent cell death. However, glycolytic generation of ATP with fructose effectively increased cellular ATP and preserved syncytial viability. These data suggest that expression of FMG in hepatoma cells results in the formation of multinucleated syncytia, causing mitochondrial failure with ATP depletion, a bioenergetic form of cell death with necrosis. This form of cell death should be effective in vivo and enhance the bystander effect, suggesting that FMG-based gene therapy deserves further study for the treatment of hepatocellular and other cancers.