The increase in UV-B radiation reaching the earth’s surface has prompted extensive studies on the effects of UV-B on plants. However, most of these studies have not addressed the close characteristics related to future survival of plant populations. The purpose of this study was to investigate the effects of UV-B radiation on reactive oxygen species (ROS) accumulation and antioxidant defense system in relation to germination, tube length and viability of maize pollen. Our results indicate that increased UV-B radiation decreased the pollen germination rate and tube length in vitro and also its fertilization ability in the field. Production of O2•− and H2O2 increased by UV-B radiation treatment, and their continuous accumulation resulted in lipid peroxidization. The activities of superoxide dismutase, catalase, peroxidase and DPPH-radical scavenging were decreased by increased UV-B radiation. The increased ROS and lipid peroxidization, and decreased activities of the antioxidants may be attributed to the effects of UV-B radiation on pollen germination, tube growth and fertilization ability.