The study aimed to determine the sterilization effect of a combination of high-pressure thermal sterilization (HPTS) and e-polylysine (PL) on Bacillus subtilis spores. The spores were treated with HPTS (550 MPa at 25°C, 65°C and 75°C) combining with e-PL at 0.1% and 0.3%. The results showed that HPTS and e-PL synergistically inactivated the spores. The increased temperature and e-PL concentration decreased the number of surviving spores, with the maximal inactivation of the spores in the treatment of 550 MPa, 75°C combining with 0.3% e-PL. The increases in the temperature and e-PL concentration significantly increased the release of the intracellular components in the spore suspension, with the maximal value for the spores treated with 550 MPa, 75°C and 0.3% e-PL. The maximal fluidity and permeability of the cell inner membrane were observed in the treatment of 550 MPa, 75°C combining with 0.3% e-PL. Changes in membrane lipids were detected from 3000 to 2800 cm -1 by Fourier transform infrared spectroscopy. The results may provide new insights into the mechanism by which HPTS and e-PL synergistically sterilize Bacillus subtilis spores.