The subcritical/supercritical carbon dioxide (SS CO2) has gained considerable attention in green chemistry industry for its advantage as nontoxic, nonflammable, and inexpensive. The effects of SS CO2 treatments on aggregation and homogenization, surface charge, secondary and tertiary structure, and activity of mushroom tyrosinase in an aqueous system were investigated using a number of methods including dynamic light scattering (DLS), zeta potential measurement, circular dichroism (CD) spectropolarimeter, and spectrofluorometer. With a treatment time of 20 min, three treatment temperatures (35,45, and 55 degrees C) and four pressures (5,8,12, and 15 MPa) had been selected. The aggregation and homogenization of the globular protein particles was induced by SS CO2 as suggested by the particle size distribution (PSD) patterns that were closely related to the pressure and temperature. The surface charge of the tyrosinase decreased following the SS CO2 treatments, and its variation tendency shows a favorable consistency with that of its PSD patterns. The alpha-helix conformation in secondary structure and fluorescence intensity reflecting tertiary structure also decreased, together with the lambda(max) red-shifted with the increasing pressure. The results also indicated that SS CO2 could enhance inactivation effect of the temperature on the tyrosinase with its lowest residual activity being about 60% under the condition of 8 MPa, 55 degrees C, and 20 min treatment time. The loss in the activity of the tyrosinase was correlated to its aggregation and homogenization effect induced by SS CO2, which led to the change of surface charge as well as secondary and tertiary structure.