Replication-conditional (oncolytic) mutants of herpes simplex virus (HSV), are considered promising therapeutic alternatives for human malignancies, and chemotherapeutic adjuvants are increasingly sought to augment their efficacy. Histone deacetylase (HDAC) inhibitors are a new class of antineoplastic agents because of their potent activity in growth arrest, differentiation, and apoptotic death of cancer cells. The ability of the HDAC inhibitors to upregulate exogenous transgene expression and inhibit interferon (IFN) responses prompted our exploration of their use in improving the antitumor efficacy of oncolytic HSV. We discovered that the yield of viral progeny increased significantly when cultured glioma cells were treated with HDAC inhibitors before viral infection. Valproic acid (VPA), a commonly used antiepileptic agent with HDAC inhibitory activity, proved most effective when used to treat glioma cells before viral infection, but not concomitantly with viral infection. Pretreatment with VPA inhibited the induction of several IFN-responsive antiviral genes, augmented the transcriptional level of viral genes, and improved viral propagation, even in the presence of type I IFNs. Moreover, VPA pretreatment improved the propagation and therapeutic efficacy of oncolytic HSV in a human glioma xenograft model in vivo. These findings indicate that HDAC inhibitors can improve the efficacy of tumor virotherapies.