Depletion of the ozone layer leads to increasing UV-B radiation on the earth's surface, which may affect weeds and their responses to herbicides. However, the effect of increased UV-B radiation on weeds and the interaction of weeds and herbicides are still obscure. The objective of this study was to compare glyphosate efficacy on velvetleaf that was grown under with and without increased UV-B radiation. Leaf area, dry weight and net photosynthesis of velvetleaf seedlings were adversely affected by increased UV-B radiation. Leaf cuticle wax significantly increased by 28% under increased UV-B radiation. Glyphosate efficacy on velvetleaf, evaluated by shoot dry weight, was significantly decreased by increased UV-B radiation. Exposure to increased UV-B radiation significantly decreased 14C-glyphosate absorption from 49% to 43%, and also resulted in less 14C-glyphosate translocation out of treated leaves and less glyphosate accumulation in newly expanded leaves. The decrease in glyphosate efficacy was due to changes in absorption and distribution, which were attributed to increased cuticle wax and decreased photosynthesis caused by increased UV-B radiation. These results suggest that the responses of weeds to herbicides may be affected by increased UV-B radiation, to the extent that higher rates may be required to achieve the desired effects.