Pulsed light (PL) is a novel technology based on heating a xenon lamp with a high-voltage current to generate a broadband light emission with an important UV component. This high-voltage does not require extreme electricity consumption because it is produced thanks to the use of capacitors that store electricity from the main during relatively long times and are discharged to the lamp in very short times. The kinetics of polyphenol oxidase (PPO) inactivation by this technology was measured and some associated structural changes were investigated by fluorometry and spectrophotometry in order to gain insight into the inactivation mechanism. To this, residual activity, tryptophan fluorescence, KI fluorescence quenching and turbidity were measured. The inactivation followed the Weibull model. The decrease in tryptophan fluorescence and the increase in Stern-Volmer constant with the progress of the PL treatment indicate the unfolding of the enzyme; a constant turbidity indicates no aggregation. The Trp fluorescence phase-diagram shows no evidence of intermediates. Therefore, these results support the idea that PL inactivates PPO by modifying its tertiary structure in an all-or-none process.