Enzymatic synthesis and hydrolysis of nucleoside phosphate compounds play a key role in various biological pathways, like signal transduction, DNA synthesis and metabolism. Although these processes have been studied extensively, numerous key issues regarding the chemical pathway and atomic movements remain open for many enzymatic reactions. Here, using the Mason–Pfizer monkey retrovirus dUTPase, we study the dUTPase-catalyzed hydrolysis of dUTP, an incorrect DNA building block, to elaborate the mechanistic details at high resolution. Combining mass spectrometry analysis of the dUTPase-catalyzed reaction carried out in H₂¹⁸O and quantum mechanics/molecular mechanics (QM/MM) simulation, we show that the nucleophilic attack occurs at the α-phosphate site. Phosphorus-31 NMR spectroscopy (³¹P-NMR) analysis confirms the site of attack and shows the capability of dUTPase to cleave the dUTP analogue α,β-imido-dUTP, containing the imido linkage usually regarded to be non-hydrolyzable. We present numerous X-ray crystal structures of distinct dUTPase and nucleoside phosphate complexes, which report on the progress of the chemical reaction along the reaction coordinate. The presently used combination of diverse structural methods reveals details of the nucleophilic attack and identifies a novel enzyme–product complex structure.