The effects of a water-miscible ionic liquid, 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF(4)]), on both thermodynamics and kinetic mechanism of the horseradish peroxidase (HRP)-catalyzed oxidation of guaiacol (2-methoxyphenol) by H(2)O(2) were investigated. The ionic liquid stabilized the ground state of guaiacol by causing an 8-fold increase of K(m) from 3 to 23 mM upon the addition of 25% (v/v) [BMIM][BF(4)]. In addition, the effect of [BMIM][BF(4)] in decreasing the k(cat) value of HRP catalysis was described by a non-competitive inhibition mechanism. The value of the inhibition constant of [BMIM][BF(4)] was 2.9 M indicating that the ionic liquid plays the role of a weak non-competitive inhibitor for HRP catalysis.