The solution speciation of the ZrIV-substituted Keggin polyoxometalate (Et2NH2)8[{α-PW11O39Zr(μ-OH)(H2O)}2]·7H2O (ZrK 2:2) was fully determined under different pD, temperature, and concentration conditions. Subsequently, phosphodiester bond hydrolysis of the DNA model substrate bis(4- nitrophenyl) phosphate (BNPP) promoted by ZrK 2:2 was studied in detail. In the presence of ZrK 2:2, phosphoester bond hydrolysis in BNPP proceeded with a rate constant of kobs = (4.750.25)10–6 s–1 at pD 6.4 and 60 °C, which represented a 320-fold rate enhancement relative to the spontaneous hydrolysis of BNPP. The pD dependence of kobs exhibits a bell-shaped profile, with the fastest rate observed at pD 6.4. An activation energy (Ea) of 60.16 kJmol–1, enthalpy of activation (ΔH#) of 57.44 kJmol–1, entropy of activation (ΔS#) of –173.16 Jmol–1K–1, and Gibbs activation energy (ΔG#) of 111.12 kJmol–1 at 37 °C were calculated. The influence of the concentration of ZrK 2:2 on the reaction rate constant was studied in the concentration range 0.5 to 6.0 mM. The results showed that ZrK 2:2 is able to hydrolyze an excess amount of BNPP, thus demonstrating that ZrK 2:2 acts as a catalyst for phospho(di)ester bond hydrolysis. In addition, the influence of ionic strength and the inhibitor diphenyl phosphate on BNPP hydrolysis were examined.