Atherosclerosis (AS) is a global disease that causes a heavy economic burden and can significantly impact human health. Pinolenic acid (PLA) has antioxidant, anti-inflammatory, and lipid-lowering effects. However, it is unclear whether PLA holds a therapeutic promise for AS treatment or prevention. This study aims to investigate whether PLA can effectively treat AS and elucidate its therapeutic mechanism. To this end, potential PLA targets in AS treatment were identified by using network pharmacology. Additionally, an in vitro AS cell model was established by H2O2-induced damaging of human coronary artery endothelial cells (HCAECs). Subsequently, endothelial cell function was evaluated by evaluating cell proliferation, oxidative markers, reactive oxygen species (ROS), and nitric oxide (NO) levels. Cellular metabolomics was further employed to assess differential intracellular metabolites following H2O2 injury. In total, 87 overlapping target genes for PLA and AS were detected. PPI network analysis identified 38 hub genes closely associated with oxidative stress and fatty acid metabolism. Moreover, PLA improved cell survival and reduced oxidative stress injury by activating the NRF2/ARE signaling pathway in vitro. Cellular metabolomics confirmed that PLA might help maintain redox homeostasis and reduce endothelial cell injury by upregulating fatty acid β-oxidation. Taken together, our findings suggest that PLA prevents H2O2-induced HCAEC injury by maintaining redox homeostasis and may, therefore, represent a therapeutic potential for AS.