Interferons (IFNs) are the most potent mediators of antiviral activities in the immune system. Viral molecular patterns are detected by receptors of the innate immune system, resulting in IFN production and subsequent IFN-stimulated gene (ISG) expression. The production of IFN serves as a danger signal that triggers a cascade of molecular events in the cell for the induction of antiviral mechanisms. The ISG products protect cells against viral replication, induce anti-proliferative mechanisms, and modulate the development of innate and adaptive immunity. The expression of ISG54 is mediated by type I IFN signaling and results in inducement of apoptosis via a mitochondrial pathway. To understand the molecular mechanism underlying this activity, we aimed to identify molecular partners of the gene product, p54. Two antioxidant enzymes were identified from a yeast two-hybrid assay, peroxiredoxin 5 (PRDX5) and superoxide dismutase 2 (SOD2). To confirm the interaction between p54 and both proteins, we cloned the cDNA of both genes and confirmed their interaction with p54 by co-immunoprecipitation and Western blot. In addition, we tested if these antioxidants could regulate ISG54-mediated apoptosis. Apoptosis was assessed using flow cytometric analysis of cells co-transfected with ISG54 with or without PRDX5 and SOD2 and co-stained with Annexin-V and propodium iodide (PI). In this report we show that both enzymes can transiently inhibit ISG54-mediated apoptosis. The results describe novel findings in the molecular mechanism of ISG54 cellular activity.