Endothelial nitric oxide synthase (eNOS), a constitutive enzyme expressed in vascular endothelial cells, is the main source of nitric oxide (NO), which plays key roles in diverse biological functions, including regulation of vascular tone. Exposure to radiation has been known to generate nitric oxide from eNOS; however, the precise mechanism of its generation and function is not known. The goal of this study was to determine the involvement of radiation-induced DNA damage response (DDR) on eNOS transcription and its effect on cell survival after irradiation. Irradiated bovine aortic endothelial cells showed increased eNOS transcription and NO generation through upregulation of ataxia-telangiectasia mutated (ATM) kinase. Radiation exposure induced NO inhibited cell death, as well as induced cellular senescence postirradiation. This study established that radiation-induced DDR uses ATM kinase to upregulate eNOS transcription and NO generation, leading to cellular senescence, which may play a critical role in radiation-mediated cardiovascular injury.