In the present study, we investigated the ability of replication-restricted herpes simplex virus (HSV) 1716 lacking ICP34.5 to infect endothelium and disrupt tumor vasculature. HSV-1716 efficiently infected and killed mouse endothelial cell lines H5V and MS1 cells, as well as human umbilical vein endothelial cells in vitro. Capillary tube formation by endothelial cells was inhibited by HSV-1716 in vitro and in vivo. Following intratumoral administration of oncolytic HSV-1716, HSV-glycoproteins could be detected in CD31-positive tumor vascular endothelium by immunostaining. Viral DNA was recovered from highly purified microdissected tumor vascular endothelium. Furthermore, endothelium of tumors treated with HSV-1716 exhibited expression of tissue factor, a marker of endothelial damage. Importantly, HSV antigen and DNA were also detected in endothelium distant from foci of active tumor infection. After intravascular inoculation of HSV-1716, viral glycoproteins were detected in association to tumor endothelium, but not vascular endothelium of different organs. Purified tumor endothelial cells showed high proliferative capability and were susceptible to HSV-1716 infection and killing ex vivo while endothelium from normal organs was not. We conclude that oncolytic HSV-1716 exerts direct antiangiogenic effects, which may contribute to the overall therapeutic efficacy of the virus.