The catalytic hydrolysis of a phosphoester bond in the DNA-model substrate 4-nitrophenyl phosphate (NPP) promoted by Zr(IV)-substituted Wells–Dawson Na14[Zr4(P2W16O59)2(μ3-O)2(OH)2(H2O)4]·57H2O polyoxometalate (ZrWD 4 : 2) was followed by means of 1H and 31P NMR spectroscopy. The hydrolytic reaction proceeded with a rate constant of 8.44 (±0.36) × 10−5 s−1 at pD 6.4 and 50 °C, representing a 300-fold rate enhancement in comparison with the spontaneous hydrolysis of NPP (kobs = 2.81 (±0.25) × 10−7 s−1) under the same reaction conditions. The ZrWD 4 : 2 was also active towards hydrolysis of bis(4- nitrophenyl) phosphate (BNPP) and the RNA model substrate 2-hydroxypropyl-4-nitrophenyl phosphate (HPNP). The pD dependence of kobs shows that the rate constants for NPP hydrolysis decrease significantly when the pD values of the reaction mixtures increase. The formation constant (Kf = 190 M−1) and catalytic rate constant (kc = 6.40 × 10−4 s−1) for the NPP-ZrWD 4 : 2 complex, activation energy (Ea) of 110.15 ± 7.06 kJ mol−1, enthalpy of activation (ΔH‡) of 109.03 ± 6.86 kJ mol−1, entropy of activation (ΔS‡) of 15.20 ± 2.49 J mol−1 K−1, and Gibbs activation energy (ΔG‡) of 104.32 ± 6.09 kJ mol−1 at 37 °C were calculated from kinetic studies. The recyclability of ZrWD 4 : 2 was examined by adding an extra amount (5.0 mM) of NPP twice to a fully hydrolyzed mixture of 5.0 mM NPP and 1.0 mM ZrWD 4 : 2. The interaction between ZrWD 4 : 2 and the P–O bond of NPP was evidenced by a change in the 31P chemical shift of the 31P atom in NPP upon addition of ZrWD 4 : 2. Based on 31P NMR experiments and the kinetic studies, a mechanism for NPP hydrolysis promoted by ZrWD 4 : 2 has been proposed.