The ionic liquid, 1-butyl-3-methylimidazolium methylsulfate ([BMIM][MeSO4]), was used to investigate the catalytic mechanism of horseradish peroxidase (HRP). The ionic liquid decreased both K-m and k(cat) values for the HRP-catalyzed oxidation of guaiacol (2-methoxyphenol) by H2O2. These studies imply that [BMIM][MeSO4] inhibits the enzyme in an uncompetitive manner. The incorporation of substrate stabilization effects measured by a thermodynamic method into the partial uncompetitive inhibition scheme successfully describes HRP-catalysis in the presence of [BMIM][MeSO4], which participates as the inhibitor. The inhibition constant of the ionic liquid was 0.051 M. The turn-over number of the native HRP was almost 14-times higher than that of the HRP-ionic liquid complex indicating that [BMIM][MeSO4] does not form a dead-end complex with HRP.