In order to explore the mechanism of HPTS in inactivation of Bacillus subtilis spores. In this paper， HPTS (200 MPa combined with 25 ℃， 65 ℃， 75 ℃； 550 MPa combined with 25 ℃， 65 ℃， 75 ℃； holding time: 20 min) was used to treat Bacillus subtilis spores. The survival concentration， UV absorption leakage， inner membrane permeability and spore composition of HPTS before and after HPTS treatment were studied by plate technology， UV spectrophotometry， flow cytometry and infrared spectroscopy. The results showed that after treated with HPTS， the survival concentration of spores decreased significantly (P<0.05)， the leakage of ultraviolet absorption increased， and the endospore membrane was damaged， resulting in the enhancement of the permeability of spore endomembrane. The results of Fourier transform infrared spectroscopy (FTIR) showed that the main differences of Bacillus subtilis spores before and after HPTS treatment were protein， lipid， nucleic acid and polysaccharide. The secondary structure of protein changed from order to disorder， and the stability of protein decreased. Therefore， HPTS can effectively destroy the permeability barrier of water molecules， lipid， protein， nucleic acid and polysaccharide structure of spore membrane， so as to reduce the resistance of spores， affect the metabolism of spores and inactivate the spores. It provides a theoretical basis for HPTS to inactivate Bacillus subtilis spores and promotes the successful application of HPTS in food processing.