This study aimed to investigate the inactivation effect of HPTS combining with egg white lysozyme on the spores of Bacillus subtilis. The spores were treated with pressure 200 MPa at 25， 65 ℃ and 75 ℃， and egg white lysozyme at 0.05%， 0.1% and 0.3%. After treatment， the inactivation rate of Bacillus subtilis spores， leakage of nucleic acid and protein， the change in the cell membrane ATPase activity， the changes in inner membrane permeability of spores and the damage on protein of the spores were determined. The results showed that HPTS and egg white lysozyme synergistically inactivated the spores， and the survival concentration of spores decreased by 3.91 lg (CFU/mL) after 200 MPa/75 ℃ combining with 0.3% lysozyme treatment. HPTS combining with lysozyme treatment significantly increased the protein and nucleic acid leakage and conductivity of the spores， significantly decreased the Na+/K+-ATPase activity， decreased the stability of the budding spore proteins， and significant increases in the permeability of the inner membrane of the spores. In summary， HPTS combining with egg white lysozyme treatment inhibited the activity of key enzymes in the spore metabolic pathway， while disrupting the spore wall， inner membrane and other structures of Bacillus subtilis budding spores， changed the physiological properties of spores， and eventually led to spore inactivation.