The effect of intense pulsed light (IPL) irradiation on Chromobacteriwn viscosum lipase was investigated with a primary focus on catalytic activity and molecular structure. During IPL irradiation, lipase activity decreased significantly with increasing pulse fluence (F-p) and exposure time (t(e)). IPL-induced deactivation kinetics were further elucidated based on a two-step series-type deactivation model (constant deactivation rate k(1) > k(2)). F-P was found to be the dominant variable affecting the degree of lipase deactivation, and residual activity was not associated with increasing to below a certain F-P energy density (2.66 mJ/cm(2)), implying a critical threshold for IPL-induced deactivation of lipase. From the results of fluorescence spectroscopy and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), we determined that IPL-induced deactivation was caused by fragmentation, leading to lipase tertiary structural changes. Furthermore, the results of FindPept analysis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) indicated that the internal sensitive bonds of lipase were cleaved preferentially by IPL, such that IPL irradiation induced site sensitive fragmentation and peptide bond cleavage.