TANK-binding kinase 1 (TBK1) is a key component of Toll-like receptor-dependent and -independent signaling pathways. In response to microbial components, TBK1 activates interferon regulatory factor 3 (IRF3) and cytokine expression. Here we show that TBK1 is a novel target of the gamma(1)34.5 protein, a virulence factor whose expression is regulated in a temporal fashion. Remarkably, the gamma(1)34.5 protein is required to inhibit IRF3 phosphorylation, nuclear translocation, and the induction of antiviral genes in infected cells. When expressed in mammalian cells, the gamma(1)34.5 protein forms complexes with TBK1 and disrupts the interaction of TBK1 and IRF3, which prevents the induction of interferon and interferon-stimulated gene promoters. Down-regulation of TBK1 requires the amino-terminal domain. In addition, unlike wild type virus, a herpes simplex virus mutant lacking gamma(1)34.5 replicates efficiently in TBK1(-/-) cells but not in TBK1(+/+) cells. Addition of exogenous interferon restores the antiviral activity in both TBK1(-/-) and TBK(+/+) cells. Hence, control of TBK1-mediated cell signaling by the gamma(1)34.5 protein contributes to herpes simplex virus infection. These results reveal that TBK1 plays a pivotal role in limiting replication of a DNA virus.