The protective effect of D-fructose on spores of Bacillus subtilis treated by high-pressure thermal sterilization (HPTS) was studied. The treatment pressure of HPTS was 200 and 600MPa, the treatment temperatures were 25,65, and 75℃, the treatment time was 20min, and the mass fractions of D-fructose were 0,15%, 30%, 60%. At these conditions, flow cytometry was used to detect the damage of spore inner membrane before and after treatment, and Fourier infrared spectroscopy was used to analyze spore protein and inner membrane lipids before and after treatment to reveal the changes in protein structure and inner membrane lipid phase. After HPTS treatment, the permeability of spore inner membrane increased and the integrity of spore inner membrane was damaged. However, with the increase of D-fructose mass fraction, the phase state of spore inner membrane lipids gradually changed from liquid crystal state to gel state, stability of inner membrane increased, permeability decreased, integrity improved, and the content of ordered secondary structure of spore protein increased. When D-fructose was not added, after treatment at 600MPa and 75℃ for 20min, the 5.31 lg(CFU/mL) spores died,and the OD₆₀₀ of spore suspension decreased to 0.41, the leakage rate of 2,6-pyridinedicarboxylic acid (DPA) reached 93.56%, the proportion of spores with inner membrane damage reached 80.95%, and the content of α-helix and β-fold structures in spore proteins decreased to 16% and 17%, respectively. When the mass fraction of D-fructose increased to 60%, the number of viable spores increased by 1.69 lg(CFU/mL) under the same HPTS treatment conditions, the OD₆₀₀ of spore suspension increased by 0.09, the proportion of spores with inner membrane damage decreased by 20.74%, and the contents of α-helix and β-fold structures of spore proteins increased to 25% and 28%, respectively. D-fructose protected the spore structure and key substances in spores during HPTS treatment, and this protective effect was positively correlated with the mass fraction of D-fructose.