Abstract Alterations of molecular properties of lipoxygenase (LOX) induced by dense phase carbon dioxide (DPCD) were analyzed using transmission electronic microscope (TEM), SDS-PAGE and native-PAGE, far UV-circular dichroism (CD) and fluorescence spectroscopy. The aggregation of LOX molecules was suggested by an increase in the particle size of LOX after DPCD treatment. The absence of LOX band was observed after exposure to DPCD at 55 °C for 30 min on the native-PAGE due to the aggregation, but the electrophoretic behavior of LOX was not altered on the SDS-PAGE. The CD and fluorescence spectra of DPCD-treated LOX were noticeably changed, its α-helix relative content decreased sharply to less than 10%, and its intrinsic relative fluorescence intensity (RFI) decreased linearly with increasing pressures. Industrial relevance As a novel non-thermal technology, DPCD can retain the quality of foods to be processed. However, the inactivation mechanism of food enzymes by DPCD is not elucidated until today. This investigation is beneficial to understand the mechanism and to push the application of this technology in food industry.