A calixarene derivative 2-[Cu(II)]2 functionalized with two cis-diaqua Cu(II) centers at the distal positions of the upper rim was synthesized and investigated as a model for dinuclear metalloenzymes that catalyze chemical transformations of phosphate esters. The flexible dinuclear calixarene efficiently catalyzes the transesterification of the RNA model 2-hydroxypropyl-p-nitrophenyl phosphate (HPNP) and the hydrolysis of the DNA model ethyl-p-nitrophenyl phosphate (EPNP) with turnover conversion, thereby exhibiting rate enhancement factors of 1.0 × 104 and 2.7 × 104, respectively. The mononuclear reference complex, 3-Cu(II), lacking the macrocyclic backbone, has a much lower activity, showing that the high catalytic activity of the dinuclear calixarene complex is due to synergetic action of the two Cu(II) centers. Saturation kinetics and pH variation studies point to the formation of a Michaelis-Menten complex in which the phosphate group is doubly Lewis acid activated by coordination to the two Cu(II) centers. In this complex, a Cu(II) bound hydroxide ion, which is present already at pH 6.5, can act as a base in the intramolecular transesterification of HPNP or as a nucleophile in the hydrolysis of EPNP. The remarkably low pKa of the Cu(II) bound water molecules in the hydrophobic calixarene 2-[Cu(II)]2 mimics the low pKa of metal bound water molecules in hydrophobic enzyme active sites, which makes the enzyme (model) active under slightly acidic to neutral conditions. The high catalytic efficiency of this enzyme model is attributed to a dynamic binding of the substrate and (pre)transition state, possible by rapid low energy conformational changes of the flexible calixarene backbone.