Methylophiopogonanone B (MO-B), which belongs to a group of homoisoflavonoids, present in Ophiopogon japonicus, has been identified as an active component with antioxidative and anti-tumor properties. The present study investigated whether MO-B may exert protective effects on human umbilical vein endothelial cells (HUVECs) against H2O2-induced injury in vitro, and whether the MO-B effects may be modulated by the NADPH pathway. HUVECs were treated with MO-B in the presence or absence of H2O2. Malondialdehyde (MDA), reactive oxygen species (ROS) levels, and superoxide dismutase (SOD) activity were analyzed to evaluate cell injury and the antioxidative potential of MO-B. The results revealed that MO-B inhibited the production of MDA and ROS, but enhanced SOD activity. Furthermore, MO-B could alleviate H2O2-induced apoptosis in HUVECs, which is consistent with the expression of apoptosis-associated genes and proteins in cells, including Bax/Bcl-2 and caspase-3. To explore the potential mechanism, the present study investigated the effects of MO-B on NADPH-related signaling via the analysis of neutrophil cytochrome b light chain (p22phox) expression, which is the membrane-associated subunit of NADPH oxidase. MO-B could improve the survival of endothelial cells and therefore may be a potential drug in the treatment of cardiovascular diseases.