The purpose of this study was to investigate the antioxidant potential of umbelliferone, 7-hydroxy coumarin, and its role in the protection against radiation-induced oxidative damage in cultured human blood lymphocytes. It was found that the antioxidant effect of umbelliferone was dose dependent in hydroxyl (OH•), superoxide anion (O2•-), 2,2-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid radical cation (ABTS•+) and 1,1-diphenyl-2-picrylhydrazyl (DPPH•) radical scavenging assays. To explore the radioprotective effect of umbelliferone, freshly isolated human blood lymphocytes were treated with 124 μM umbelliferone (optimum dose-fixed by MTT assay) 30 min before 3 Gy irradiation. It was found that umbelliferone pretreatment inhibited radiation-induced reactive oxygen species generation in 3 Gy exposed lymphocytes. Microscopic observations showed that there was a significant apoptotic cells (ethidium bromide/acridine orange staining) and decreased mitochondrial membrane potential (Rhodamine 123 staining) in irradiated lymphocytes. On the other hand, 124 μM umbelliferone treatment significantly decreased % of apoptotic cells and prevented radiation induced mitochondrial depolarization in lymphocytes. Further, it was noticed that there was an increased DNA damage (comet assay), lipid peroxidation with decreased antioxidant enzymatic i.e., superoxide dismutase, catalase and, glutathione peroxidase activities in 3 Gy irradiated lymphocytes. Conversely, umbelliferone (124 μM) treatment before irradiation decreased comet attributes and lipid peroxidative markers with improved antioxidant enzyme activities in irradiated lymphocytes. Taken together, the results of this study clearly suggest the radioprotective effect of umbelliferone in human lymphocytes by inhibiting reactive oxygen species generation and its subsequent toxicity.