In this study, flavonoid rutin was acylated with lauric acid in acetone catalyzed by an immobilized lipase from Candida antarctica. In addition to the reaction not being well studied, the esterification also involves two immiscible substrates, which posed a significant challenge. Analysis indicated that the lipase-mediated esterification exhibits a ping-pong bibi mechanism with no apparent inhibition by the two substrates. The activation energy, E-a, for the esterification was calculated as similar to 37 kJ mol1. The esterification process was found to be endothermic, with enthalpy (Delta H) and entropy (Delta S) values of approximately +50 kJ mol(-1) and +110 J mol(-1) K-1, respectively. Based on the value of the Gibbs free energy change (Delta G), the esterification reaction under the conditions studied was predicted to be nonspontaneous below 175 degrees C but spontaneous at higher temperatures. The study also confirmed, through C-13 NMR analysis, the exact location of rutin esterification. The investigated esterification biochemistry highlighted important behavior concerning the enzyme-mediated synthesis of functionalized flavonoid.