15-Lipoxygenases (15-LOs) catalyze the peroxidation reaction of linoleic acid (LA) in mammals producing almost exclusively 13-(S)-hydroperoxyoctadecadienoic acid (13-(S)-HPODE). Although several hypotheses have been formulated, the molecular basis of such enzymatic regiospecificity is unclear. We have here combined quantum mechanics/molecular mechanics (QM/MM) calculations with molecular dynamics simulations to analyze the peroxidation mechanism using a complete rabbit 15-LO-1/LA solvated model. C-9 and C-13 being equivalent for planarity and spin density, the QM/MM potential energy profiles of the O-2 addition to those two atoms were calculated. The difference in the potential energy barrier heights is clear enough to justify that O-2 selectively attacks C-13 giving 13-(S)-HPODE. Oxygenation at C-9 is hindered by two steric-shielding residues (Leu597 and Gln548). The calculated free energy profile at 300 K for the O-2 addition to C-13 confirms that the peroxidation on C-13 is a reversible viable process in agreement with experiments. Thus, the subsequent reduction of the peroxyl radical to give the final hydroperoxidated product is expected to give the irreversibility character to the overall process.