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.