In order to broaden the antimicrobial action spectrum of lysozyme against Gram-negative bacteria, different partially reduced lysozyme derivatives were obtained by reducing the R-S-S-R bonds of lysozyme using the reducing agent Na2SO3. The circular dichroism behavior analysis showed that the tertiary structure of the partially reduced lysozyme molecules became more flexible, leading to an obvious increase in the surface hydrophobicity and the tryptophan fluorescence intensity. Zeta potential analysis indicated that the introduction of SO32- led to a decrease in the surface charges of the reduced lysozyme molecules. Compared with the native lysozyme, the reduced lysozymes not only showed a 1 log increase in the antimicrobial activity against Escherichia coli ATCC 25922 and Salmonella enteritidis ATCC 13076 (P <0.05), but also maintained nearly the same antimicrobial activity against Staphylococcus aureus ATCC 29213 (P >0.05). Overall, the reductive modification with the food-friendly, compatible, and safe reducing agent Na2SO3 has broadened the antimicrobial action spectrum of the modified lysozymes against Gram-negative bacteria, with the enhancement extent depending on the reduction degree and the type of bacterial strains. The integrated results suggest that the Na2SO3-reduced lysozyme can be used as a novel safe potential bactericidal additive for food-processing industry.