The ability of adenovirus early region proteins, E1B-55K and E4orf6, to usurp control of cellular ubiquitin ligases and target proteins for proteasome-dependent degradation during infection is well established. Here we show that the E4 gene product, E4orf3 can, independently of E1B-55K and E4orf6, target the transcriptional corepressor transcriptional intermediary factor 1 gamma (TIF1 gamma) for proteasome-mediated degradation during infection. Initial mass spectrometric studies identified TIF1 family members-TIF1 alpha, TIF1 beta, and TIF1 gamma-as E1B-55K-binding proteins in both transformed and infected cells, but analyses revealed that, akin to TIF1 alpha, TIF1 gamma is reorganized in an E4orf3-dependent manner to promyelocytic leukemia protein-containing nuclear tracks during infection. The use of a number of different adenovirus early region mutants identified the specific and sole requirement for E4orf3 in mediating TIF1 gamma degradation. Further analyses revealed that TIF1 gamma is targeted for degradation by a number of divergent human adenoviruses, suggesting that the ability of E4orf3 to regulate TIF1 gamma expression is evolutionarily conserved. We also determined that E4orf3 does not utilize the Cullin-based ubiquitin ligases, CRL2 and CRL5, or the TIF1 alpha ubiquitin ligase in order to promote TIF1 gamma degradation. Further studies suggested that TIF1 gamma possesses antiviral activity and limits adenovirus early and late gene product expression during infection. Indeed, TIF1 gamma knockdown accelerates the adenovirus-mediated degradation of MRE11, while TIF1 gamma overexpression delays the adenovirus-mediated degradation of MRE11. Taken together, these studies have identified novel adenovirus targets and have established a new role for the E4orf3 protein during infection.