Viral microRNAs are one component of the RNA interference phenomenon generated during viral infection. They were first identified in the Herpesviridae family, where they were found to regulate viral mRNA translation. In addition, prior work has suggested that Kaposi's sarcoma-associated herpesvirus (KSHV) is capable of regulating cellular gene transcription by miRNA. We demonstrate that a miRNA, hsv1-mir-H27, encoded within the genome of herpes simplex virus 1 (HSV-1), targets the mRNA of the cellular transcriptional repressor Kelch-like 24 (KLHL24) that inhibits transcriptional efficiency of viral immediate-early and early genes. The viral miRNA is able to block the expression of KLHL24 in cells infected by HSV-1. Our discovery reveals an effective viral strategy for evading host cell defenses and supporting the efficient replication and proliferation of HSV-1.