The enzyme human aromatase (HA) catalyzes the conversion of androgens to estrogens via two hydroxylation reactions and a final unique aromatization step. Despite the great interest of HA as a drug target against breast cancer detailed structural and spectroscopic information on this enzyme became available only in the past few years. As such, the enigmatic mechanism of the final aromatization step is still a matter of debate. Here, we investigated the final step of the HA enzymatic cycle via hybrid quantum-classical (QM/MM) metadynamics and blue-moon ensemble simulations. Our results show that the rate-determining step of the aromatization process is the nucleophilic attack of the distal oxygen of a peroxo-ferric species on the formyl carbon of the enol-19-oxo-androstenedione, which occurs with a free energy barrier (ΔF(#)) of ∼ 16.7 ± 1.9 kcal/mol, in good agreement with experimental data. This reaction is followed by a water mediated 1β-hydrogen abstraction (ΔF(#) = 7.9 ± 0.8 kcal/mol) and by the formation of a hydroxo-ferric moiety. This latter may be finally protonated by a hydrogen delivery channel involving Asp309 and Thr310, both residues pointed out as crucial for HA activity. In the absence of the catalytic water in the active site the substrate does not assume a position suitable to undergo the nucleophilic attack. Our data not only reveal a novel possible mechanism for the aromatization process consistent with some of the spectroscopic and kinetic data available in the literature, complementing current knowledge on the mechanism of this enzyme, but also point out a remarkable influence of the level of theory used on the calculated free energy barriers. The structural information obtained in this study may be used for the rational structure-based drug design of HA inhibitors to be employed in breast cancer therapy.