Polo-Like Kinase (PLK) inhibitors, such as BI-2536, have been reported to suppress interferon β (IFNβ) gene transcription induced by ligands that activate TLR3 and TLR4. Here, we report that BI-2536 is likely to exert this effect by preventing the interaction of the transcription factors IRF3 and c-Jun with the IFNβ promoter, but without affecting the TANK-binding kinase 1 (TBK1)-catalysed phosphorylation of IRF3 at Ser396, the dimerization and nuclear translocation of IRF3 or the phosphorylation of c-Jun and ATF2. Although BI-2536 inhibits few other kinases tested, it interacts with Bromodomain and Extra-Terminal (BET) family members and displaces them from acetylated lysine residues on histones. We found that BET inhibitors that do not inhibit PLKs, phenocopied the effect of BI-2536 on IFNβ gene transcription. Similarly, BET inhibitors blocked the interaction of IRF5 with the IFNβ promoter and the secretion of IFNβ induced by TLR7 or TLR9 ligands in the human plasmacytoid dendritic cell line GEN2.2, but without affecting the nuclear translocation of IRF5. We found that the BET family member BRD4 was associated with the IFNβ promoter and that this interaction was enhanced by TLR3- or TLR4-ligation and prevented by BI-2536 and other BET inhibitors. Our results establish that BET family members are essential for TLR-stimulated IFNβ gene transcription by permitting transcription factors to interact with the IFNβ promoter. They also show that the interaction of the IFNβ promoter with BRD4 is regulated by TLR ligation and that BI-2536 is likely to suppress IFNβ gene transcription by targeting BET family members.