To elucidate the mechanism of inactivation of mushroom polyphenoloxidase (PPO) by nonthermal treatment, PPO solutions were irradiated up to 10 kGy or pressurized at 600 MPa and 800 MPa, respectively. PPO activities were significantly affected by γ irradiation, and treatment at 5 kGy decreased the activity by 93%. Treatment of PPO at 600 MPa decreased the activity slightly, yet 10 and 20 min treatments at 800 MPa decreased the activities by 28% and 43%, respectively. Circular dichroism study showed that nonthermal treatment altered the ellipticity values in the range of 210 and 225 nm, resulting in decrease of the ordered structure. Fluorescence spectroscopy indicated that nonthermal treatment quenched the emission intensity.