Interferon regulatory factor 3 (IRF3) is a key transcription factor involved in the induction of interferon (IFN) in response to viral infection. Rotavirus non-structural protein NSP1 binds to and targets IRF3 for proteasome degradation early post-infection. Mutational analysis of cysteine and histidine residues within the conserved N-terminal zinc-binding domain in NSP1 of bovine rotavirus strain B641 abolished IRF3 degradation in transfected cells. Thus, the integrity of the zinc-binding domain in NSP1 is important for degradation of IRF3. In contrast to bovine strain B641, IRF3 was stable in cells infected with porcine rotavirus strain OSU and OSU NSP1 bound only weakly to IRF3. Both B641 NSP1 and OSU NSP1 were stabilized in cells or cell-free extracts in the presence of the proteasome inhibitor MG132 and when the zinc-binding domain was disrupted by site-directed mutagenesis. Data from the B641 analyses that show IRF3 degradation is dependent on the presence of NSP1 and the integrity of the N-terminal zinc-binding domain, coupled with the regulated stability of IRF3 and NSP1 by the proteasome, collectively support the hypothesis that NSP1 is an E3 ubiquitin ligase.