Interferon Regulatory Factor (IRF-3) has been shown to contribute to immune control of B16 melanoma tumor growth. We have shown previously that IRF-3 has a role in IFN-γ-induced expression of pro-apoptotic interferon stimulated gene 54 (ISG54) in macrophages and IFN-γ in T cells. To investigate the IRF3-IFN-γ-ISG54 nexus, we injected C57Bl/6 (B6) and IRF3KO mice s.c. with luciferase-producing B16-F10 tumor cells. Tumor growth as measured by luciferase levels was similar between B6 and IRF3KO mice at days 2 and 6, but was significantly greater at day 9 in IRF3KO mice compared with B6 mice. Transcription factor assays on splenic protein extracts after tumor inoculation revealed peak activation of IRF3 and IRF7 at day 6 in B6 tumor-bearing mice but not in IRF3KO tumor-bearing mice. Likewise, significant induction of IFN-γ occurred in spleens and tumors in B6 mice from days 6–9 but failed to occur in tumor-bearing IRF3KO mice. Previous reports from other labs showed that the anti-tumor properties of IFN-γ are the result of cell cycle arrest. Using B16F1 cells or B16F1 cells deficient in IFN-γ receptor (B16-IRFGRKO), we found that IFN-γ alone and in synergy with the TLR3/IRF3 agonists, poly I:C, decreased B16F1 cell growth in significant correlation with increased ISG54 expression. Moreover, IFN-γ alone increased expression of the cell cycle inhibitor, p27Kip while IFN-γ plus poly I:C increased cleaved Caspase-3 in B16 cells. Thus, it is likely that an IFN-γ/IRF3/ISG54 nexus can significantly contribute to tumor cell control during anti-tumor immune responses.