HSV-1 ICP27, an immediate early protein of herpes simplex virus type 1, is involved in viral replication, transcriptional activation, RNA stability, apoptosis, and reactivation from viral latency. The reactivation from viral latency is closely related to apoptosis and oxidative stress. To understand the effect of ICP27 upon apoptosis, we tested for cell death of ICP27-expressing cells (3-3) in the presence of hydrogen peroxide. Under oxidative stress, 3-3 cells were sensitive to death, displaying typical apoptosis features such as oligonucleosomal DNA fragmentation, chromatin condensation, and G0/G1 accumulation. To dissect the sensitizing mechanism of ICP27 in apoptosis, we analyzed the level of intracellular reactive oxygen species (ROS) in 3-3 cells. We found that 3-3 cells exhibited increased ROS levels compared to Vero cells devoid of ICP27. We also observed that the increased levels of intracellular ROS in 3-3 cells were caused by disturbances in antioxidant enzymes. In 3-3 cells, the elevated ROS increased the AP-1 activity, subsequently the expression of Bax increased, and the expression of Bcl2 was reduced. In addition, 3-3 cells were sensitive to various apoptotic agents. Taken together, these results indicate that HSV-1 ICP27 sensitizes the cells to apoptosis by elevating the intracellular levels of ROS.