We have isolated and characterized a novel variant of the replication-competent oncolytic HSV1716 that expresses inhibitor of growth 4 (Ing4) (HSV1716Ing4). We demonstrate that Ing4 expression enhances progeny output during HSV1716 infection of human tumor cells both in vitro and in vivo, thereby significantly augmenting its oncolytic potency. In tissue culture, compared with HSV1716, HSV1716Ing4 produced significantly higher numbers of infectious progeny in human squamous cell carcinoma (SCC), breast, ovarian, prostate and colorectal cancer cell lines. Immediate-early expression of Ing4 was crucial for this effect and an intact Ing4 was required as there was no enhanced progeny production with HSV1716 variants that expressed Ing4 mutants lacking the C-terminal plant homeodomain domain or conserved nuclear localization signals. In mouse xenograft models of SCC, ovarian and breast cancer, HSV1716Ing4 was significantly more efficacious than HSV1716 with at least 1000-fold more infectious virus found in tumors after HSV1716Ing4 treatment compared with tumors from HSV1716 treatment. Using a sensitive herpes simplex virus type 1 (HSV-1) PCR, virus DNA was only detected in tumors and was not detected in the DNA extracted from any organs of the injected mice demonstrating that, like HSV1716, HSV1716Ing4 replication is exclusively restricted to tumor cells. Our results suggest that the potential for enhanced tumor destruction by oncolytic HSV expressing Ing4 merits clinical investigation.