The inactivation mechanism of PMF on Listeria grayi (L. grayi) was investigated through the analysis of biological effects as well as the monitoring of morphology, membrane permeability and intracellular proteins of cells. Under the optimal inactivation conditions of PMF (2.5 T and 25 pulses), the analysis of transmission electron microscopy (TEM) and laser scanning confocal microscopy (LSCM) showed that PMF‐treated cells membranes were damaged, resulting in the increase in intracellular Ca2+ fluorescence intensities. A significant (P < 0.01) negative correlation between intracellular Ca2+ fluorescence intensities and the amount of colonies was found. Proteomic analysis showed that the mode‐of‐action of cells outer membrane, the stability of intracellular proteins and the metabolism‐related proteins might be affected by PMF. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that PMF treatment could affect the nitrogen, organic and energy metabolisms of L. grayi, inducing the death of cells finally.