The reaction mechanism of human deoxyribonucleotidase (dN) is studied using high-level quantum-chemical methods. dN catalyzes the dephosphorylation of deoxyribonucleoside monophosphates (dNMPs) to their nucleoside form in human cells. Large quantum models are employed (99 atoms) based on a recent X-ray crystal structure [Rinaldo-Matthis et al. Nat. Struct. Biol. 2002, 9, 779]. The calculations support the proposed mechanism in which Asp41 performs a nucleophilic attack on the phosphate to form a phospho-enzyme intermediate. Asp43 acts in the first step as an acid, protonating the leaving nucleoside, and in the second step as a base, deprotonating the lytic water. No pentacoordinated intermediates could be located.