Abstract: A systematic investigation was implemented to clarify the inactivation effect and mechanism of combining heat and lysozyme treatment on Bacillus subtilis spores. The spores were treated by 80°C/90°C combined with 0.2%/0.4% lysozyme for 30 min. The combined treatment of heat and lysozyme exhibited a synergistic effect on spore inactivation. There was a significant sporicidal effect in treatment with lysozyme alone. Once combined with heat treatment at 80 °C, 0.2% and 0.4% lysozyme achieved spore inactivation efficiencies of (1.17±0.25) lg CFU/mL and (1.57±0.12) lg CFU/mL, respectively. The inactivation effect reached(3.19±0.49) lg CFU/mL after treatment with 90℃-0.4% lysozyme for 30 min. D- and Z-values of the spores decreased after treatment with heat and lysozyme, indicating that the addition of lysozyme significantly accelerated the death of Bacillus subtilis spores. D-values were reduced to 12.31 and 9.40 min, respectively, and the Z-values were 36.33 °C and 25.48 °C, respectively, under the treatments of 90 °C combined with 0.2% and 0.4% lysozyme. Laser confocal microscopy revealed that the spores were stained by SYTO16 after treatment, indicating the damage to the cortex and cell wall. Furthermore, the cortical structure of spores increased to emit green fluorescence after hydrolysis with the addition of 0.4% lysozyme at 90 ℃. Fourier transform infrared spectra (FTIR) indicated that the β-1,4 glycosidic bonds of peptidoglycan were broken in the cortex and cell wall. The relative content of free sulfhydryl groups increased significantly (5.49±0.12 μmol/g protein) under the combined treatment of 90 °C heat and 0.4% lysozyme. Peak fitting (1700–1600 cm^-¹) of the FTIR spectrum indicated the alterations in the spore protein secondary structure. The relative contents of α-helix and β-sheet decreased markedly, while those of β-turn and random coil increased significantly. The protein conformation transformed from an ordered to a disordered state. Protein stability was reduced to disrupt the spore coat’s protein network. The lysozyme passed through the damaged coat without the barrier against lysozyme, leading to synergistic damage to the cortex and cell wall. Phase-contrast microscopy demonstrated that the inner membrane was also damaged without protective effects from the coat and cortex, leading to core hydration. The spores shifted from phase-bright to phase-dark state under a phase-contrast microscope. In summary, structural damages were also responsible for the spore inactivation under combined heat and lysozyme treatment.