Regio- and enantioselective hydroxylation of free fatty acids by human cytochrome P450 2E1 (CYP2E1) plays an important role in metabolic regulation and has significant pathological implications. Despite extensive research, the detailed hydroxylation mechanism of CYP2E1 remains incompletely understood. To clarify the origins of regioselectivity and enantioselectivity observed for CYP2E1-mediated fatty acid hydroxylation, molecular dynamics (MD) simulations and quantum mechanics/molecular mechanics (QM/MM) calculations were performed. MD simulations provided key insights into the proximity of arachidonic acid's carbon atoms to the reactive iron(IV)-oxo moiety in compound I (Cpd I), with the ω-1 position being closest, indicating higher reactivity at this site. QM/MM calculations identified hydrogen abstraction as the rate-determining step, with the ω-1