DNA replication greatly enhances expression of the Herpes simplex virus 1 (HSV-1) γ2 late genes by still unknown mechanisms. Here we demonstrate that 5,6-dichloro-1-β–D-ribofuranosylbenzimidazole (DRB), an inhibitor of CDK9, suppresses expression of γ2 late genes with an IC50 of 5 μM, which is at least ten times lower than the IC50 required for inhibition of expression of early genes. The effect of DRB could not be explained by inhibition of DNA replication per se or loading of RNA polymerase II to late promoters and subsequent reduction of transcription. Instead, DRB reduces accumulation of γ2 late mRNA in the cytoplasm. In addition, we show that siRNA mediated knock-down of the transcription factor SPT5, but not NELF-E, also gives rise to a specific inhibition of HSV-1 late gene expression. Finally, addition of DRB reduces co-immunoprecipitation of ICP27 using an anti-SPT5 antibody. Our results suggest that efficient expression of replication-dependent γ2 late genes is, at least in part, regulated by CDK9 dependent co- and/or post-transcriptional events involving SPT5 and ICP27.