Vessel endothelium injury caused by reactive oxygen species (ROS) including H2O2 plays a critical role in the pathogenesis of cardiovascular disorders. Therefore, drug targeting ROS elimination has highly clinical values in cardiovascular therapy. The plant of Radix Ophiopogon japonicus is a traditional Chinese herbal medicine that has been commonly used for prevention and treatment of cardiovascular diseases for a long history. However, the effective component mediating its beneficial effects remains unknown. In the present study, we investigated the action of Ophiopogonin D (OP-D), one of the most bioactive components of Radix Ophiopogon japonicus, in an endothelial injury model induced by H2O2. We found that OP-D inhibited mRNA levels of antioxidant, inflammatory and apoptotic genes in a dose-dependent manner. H2O2-induced lipid peroxidation and protein carbonylation were reduced by OP-D pretreatment. Mitochondrial ROS generation and cell apoptosis were also attenuated in OP-D pretreated cells. In addition, OP-D restored cellular total antioxidative capacity and inhibited the release of inflammatory cytokines. Furthermore, OP-D suppressed the enzymatic activity of Catalase, HO-1, and Caspases. Finally, OP-D blocked activation of NF-κB and ERK signaling cascades. Taken together, our findings provide the first evidence that OP-D plays a protective role as an effective antioxidant in H2O2-induced endothelial injury. Ophiopogonin D can be therefore developed as a novel drug for the therapy of cardiovascular disorders.