Walleye dermal sarcoma virus (WDSV) is a complex retrovirus that causes the growth of multifocal, cutaneous tumors in walleye fish (Sander vitreus vitreus). These virus-induced tumors spontaneously regress on a seasonal basis. The WDSV genome encodes three accessory proteins (rv-cyclin, Orf B, and Orf C) that are necessary for regulation of virus expression, tumor formation, and tumor regression. While rv-cyclin and B are critical for tumor development, Orf C contributes to the observed seasonal tumor regression. Previous studies have shown that Orf C targets the cell mitochondria and induces apoptosis. These studies suggest that Orf C-induced apoptosis leads to the observed tumor regression in fish infected with WDSV. To further define the mechanism(s) of apoptosis, we generated a recombinant lentivirus (Lenti Orf C) that expresses WDSV Orf C. By infecting cells with Lenti Orf C, we showed decreasing cell viability in association with increasing virus concentrations. We also demonstrated Orf C expression in mitochondrial, cytosolic, and nuclear cell fractions, with the strongest Orf C expression in cell nuclei. In addition, we identified two pro-apoptotic proteins that associate with Orf C, ANT and Bax, and identified a third protein, AIF, as a potential Orf C target. While significant progress has been made in elucidating the mechanism(s) of Orf C-induced apoptosis, further studies are necessary to determine which cellular proteins are the primary targets of Orf C. These apoptosis-inducing Orf C targets may be useful in developing future oncolytic therapies.