In order to explore the inactivation mechanism of electron beam irradiation against Listeria， the effects of intracellular components and related gene expression on Listeria were studied by fourier transform infrared spectroscopy and real-time fluorescence quantitative PCR. The results showed that after the electron beam irradiation， the absorption peak intensity of the intracellular components of Listeria decreased， and the absorption frequency shifted， which resulted in the changes of the structure and conformation of intracellular membrane fatty acids， proteins， nucleic acids and other substances. And in the amide I band the content of α-helix and β-turn was significantly reduced， and the content of β-sheet and random coil were significantly increased， indicating that the intracellular protein structure became disorder after irradiation. In addition， electron beam irradiation treatment resulted in the elevations of recA gene and glucose metabolism related genes， while reductions in the level of virulence factors. The above results indicated that electron beam irradiation had significant effects on the intracellular components and the related genes expression of Listeria， and these changes may play an important role in the death of L. innocua caused by electron beam irradiation.