The efficiency of low-pressure carbon dioxide microbubbles (CO2MB) to inactivate alpha-amylase was analysed kinetically, and structural alteration of alpha-amylase by CO2MB was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorescence analysis of tryptophan (Trp) residues. Activity and Trp fluorescence intensity of alpha-amylase treated by CO2MB decreased with increasing temperature, pressure and exposure time, and lowering the initial buffer pH, respectively. In the kinetic analysis, it was confirmed that the decreased temperature-dependency and increased activation energy associated with the inactivation of alpha-amylase by CO2MB were induced by pressurizing the mixing vessel and that the decreased pressure-dependency and increased activation volume concomitant to the inactivation of alpha-amylase by CO2MB was induced by increasing the temperature in the heating coil. In SDS-PAGE, CO2MB was suggested to induce the structural alteration of alpha-amylase because the band density decreased after CO2MB treatment, although this phenomenon was not related to the inactivation efficiency. However, Trp fluorescence analysis showed that the alteration of the tertiary structure of alpha-amylase by CO2MB was related to the inactivation efficiency. Therefore, CO2MB was more effective than thermal treatment in inactivating alpha-amylase, and the inactivation efficiency was suggested to be related to the alteration of the enzyme's tertiary structure.