Proton beam therapy can kill tumor cells while saving normal cells because of its specific energy delivery properties and so is used to various tumor patients. However, the effect of proton beam on angiogenesis in the development of blood vessels has not been determined. Here we used the zebrafish model to determine in vivo whether proton beam inhibits angiogenesis. Flk-1-GFP transgenic embryos irradiated with protons (35 MeV, spread out Bragg peak, SOBP) demonstrated a marked inhibition of embryonic growth and an altered fluorescent blood vessel development in the trunk region. When cells were stained with acridine orange to evaluate DNA damage, the number of green fluorescent cell death spots was increased in trunk regions of irradiated embryos compared to non-irradiated control embryos. Proton beam also significantly increased the cell death rate in human umbilical vein endothelial cells (HUVEC), but pretreatment with N-acetyl cystein (NAC), an antioxidant, reduced the proton-induced cell death rate (p<0.01). Moreover, pretreatment with NAC abrogated the inhibition of trunk vessel development and prevented the trunk malformation caused by proton irradiation. In conclusion, proton irradiation significantly inhibited in vivo vascular development possibly due to increased vascular cell death via reactive oxygen species formation.