Ferroptosis is a regulated form of cell death driven by lipid peroxidation, with 15-lipoxygenase (15LOX) enzyme playing a critical role in catalyzing the oxygenation of polyunsaturated fatty acid-containing phospholipids, such as 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine (SAPE), to initiate this process. The molecular oxygen required for this catalytic reaction is subject to continuous competition among various oxygen-consuming enzymes, which influences the efficiency of lipid peroxidation. In this study, we utilized structure-based modeling and all-atom molecular dynamics simulations to explore the oxygen diffusion pathways in 15LOX-1 under varying oxygen concentrations and in the presence of key components, including a substrate, binding partner PE-binding protein 1 (PEBP1), and the membrane environment. Extensive computational experiments were performed on various system configurations, examining the role of substrate binding, membrane presence, and PEBP1 association in oxygen acquisition. Our computational results indicate that the substrate binding induces a conformational change in 15LOX-1, facilitating the simultaneous recruitment of one or two O2 molecules, which drive peroxidation, leading predominantly to monohydroperoxide products and, less frequently, to dihydroperoxide products. A similar trend was observed in our redox lipidomics analysis. Moreover, we noticed that the presence of the membrane significantly reduces irrelevant oxygen binding spots, directing oxygen molecules toward a primary tunnel essential for the catalytic activity. We identified two primary oxygen tunnels with sequentially and structurally conserved regions across the lipoxygenase family. These findings provide novel insights into the regulation of oxygen acquisition mechanism for LOX members, shedding light on the molecular basis of ferroptosis signaling.