Pioglitazone is an important prescription antidiabetic drug with positive roles in controlling high blood sugar in patients with type 2 diabetes. In the present study, we investigated the effects of pioglitazone on the structure and function of bovine liver catalase (BLC) using different spectroscopic and theoretical methods. UV-Vis absorption, fluorescence spectroscopy, synchronous fluorescence, and circular dichroism studies revealed conformational changes in the BLC structure and heme group in the presence of different concentrations of pioglitazone. Kinetic studies indicated that pioglitazone can increase BLC activity by approximately threefold compared with free enzyme. The fluorescence quenching data showed one binding site for pioglitazone, and the binding constants at 298, 304, and 310 K were calculated as 5.01 × 107 M-1 , 5.8 × 107 M-1 , and 6.6 × 107 M-1 , respectively. The static type of quenching mechanism was mainly involved in the quenching of intrinsic emission of the enzyme. Thermodynamic data suggested that hydrophobic interactions played a major role in the binding reaction of pioglitazone with BLC. The molecular docking studies indicated that pioglitazone interacts with the cavity in the middle of the β-barrel and wrapping domain of BLC. Thus, pioglitazone can increase catalase activity by changing the BLC structure.