The present study investigated the effects of hesperetin on vessel structure formation in mouse embryonic stem (mES) cells with regard to whether hesperetin acts as an antioxidant or pro-oxidant. Some flavonoids enhance antioxidant systems while increasing oxidative stress in the body. After their differentiation into endothelial-like cells for 10 d, mES cells were treated with 1 to 100 μM of hesperetin for 24 h. Hesperetin efficiently inhibited the formation of vessel-like tubular structures consisting of platelet-endothelial cell adhesion molecule-1–immunoreactive cells and significantly (P < 0.05) increased the generation of reactive oxygen species in a concentration-dependent manner. Although glutathione (in its reduced and oxidized forms) in mES cells was not affected by hesperetin, the 8-iso-prostaglandin F2 α content was decreased. In addition, cytotoxicity-induced hesperetin was not found; lactate dehydrogenase release and cell viability were determined as an index of cell damage. Taken together, the present study shows that hesperetin inhibits vessel formation by pro-oxidant means and suggests its potential as an antiangiogenic agent.