•  The enzyme phosphatidylcholine‐specific phospholipase C (PC‐PLC) participates in atherosclerosis development and may negatively regulate autophagy. •  Phosphatidylethanolamine binding protein 1 (PEBP1) represents a novel effector of signal transduction pathways that control cellular growth, motility, apoptosis, genomic integrity, and therapeutic resistance. •  The disruption of PEBP1 was reported to associate with a wide range of diseases, such as cancer, pancreatitis, and Alzheimer's disease, making it a potential target for disease therapy. However, the roles of PEBP1 in vascular endothelial cell (VEC) autophagy and arteriosclerosis are not clear. •  Here we report that PEBP1 interacts with PC‐PLC and positively regulates PC‐PLC activity, while both PEBP1 and PC‐PLC negatively regulate VEC autophagy. •  The PEBP1 level is elevated during the development of atherosclerosis, and the PC‐PLC inhibitor D609 significantly decreases the upregulated PEBP1 level in apolipoprotein E−/− mice, suggesting that PEBP1 may be a potential diagnostic indicator for atherosclerosis. Abstract  We previously found that phosphatidylcholine‐specific phospholipase C (PC‐PLC) was a key inducing element of atherosclerosis, and might negatively regulate human umbilical vein endothelial cell (HUVEC) autophagy. To further investigate the mechanism of PC‐PLC action, we initially identified phosphatidylethanolamine binding protein 1 (PEBP1) as a binding partner of PC‐PLC by using mass spectrometry (MS, MALDI‐TOF/TOF). We found that PEBP1 positively regulated PC‐PLC activity in HUVECs, and inhibition of PC‐PLC by its inhibitor D609 suppressed PEBP1 expression dramatically. Moreover, both PC‐PLC and PEBP1 negatively regulated HUVEC autophagy independently of mammalian target of rapamycin (mTOR). Furthermore, the PEBP1 level was elevated during the development of atherosclerosis, while D609 significantly decreased the upregulated PEBP1 level in apoE−/− mice.