<p>Ferroptosis is a novel form of programmed cell death distinct from apoptosis, necrosis, and autophagy, characterized by the abnormal accumulation of iron-dependent lipid peroxides. Increasing evidence suggests that ferroptosis is widely involved in various diseases, including neurodegenerative disorders, cancers, and cardiovascular diseases. However, the mechanisms of enzymes promoting lipid peroxidation remain poorly understood. The endoplasmic reticulum monooxygenase (MMO) mainly composed of cytochrome P450 (CYPs) and cytochrome P450 reductase (CPR) transfers electrons from nicotinamide adenine dinucleotide phosphate (NADPH) to molecular oxygen, leading to the generation of reactive radical intermediates and hydrogen peroxide. Subsequently, peroxidation of unsaturated liposomes disrupts membrane integrity and leads to ferroptosis. Herein, we elucidate the molecular details of MMO-liposome interactions and reveal their association with phospholipid membrane permeabilization in ferroptosis. Using Raman spectroscopy, we found that MMO interacted with unsaturated liposomes in the presence of electron donors, disrupting the conformation order of fatty acid chains. Furthermore, the degree of unsaturation in the liposomes positively correlates with the extent of peroxidation. Simultaneously, through the Raman spectra of the CYP 3A4 redox center, the relationship between MMO-liposome interactions and reactive oxygen species (ROS) generation is revealed, providing new insights into the mechanistic analysis of MMO-mediated ferroptosis. Moreover, the addition of Fe³⁺ promotes the generation of reactive hydroxyl radicals, accelerating the extent of liposomal peroxidation. This study provides new mechanistic insights into how MMO promotes ferroptosis through lipid peroxidation and offers a theoretical basis for the development of novel anticancer therapeutic strategies.</p> Graphical abstract <p></p>