We report the characterization of VvDRT100-L, a grape DNA-damage repair/toleration 100 protein. VvDRT100-L has nine leucine-rich repeats and belongs to the plant DRT100 protein family. VvDRT100-L is expressed abundantly in green organs of grapevines, including tendrils, leaves, and green berry skins. The overexpression of VvDRT100-L in Arabidopsis plants decreased the number of abasic sites and the frequency of DNA single-strand breaks in the DNA damaged by UV-B irradiation, whereas UV-B irradiation markedly increased the number of abasic sites and the frequency of DNA single-strand breaks in T-DNA insertion mutant drt100 plants. VvDRT100-L-overexpressing plants remained viable and noticeably healthy under lethal UV doses, suggesting that VvDRT100-L may enhance UV tolerance in plant. Taken together, we concluded that VvDRT100-L might play an important role in the repair and toleration of UV-B-induced DNA damage. These findings would help us better understand how plants acquire UV stress acclimation, tolerance and DNA repair.