In order to study the optimal modification process and characteristics of natto polysaccharides, sulfonic acid-pyridine method was used to sulfate natto polysaccharides. Single factor experiments and orthogonal experiments were used to determine the optimal process conditions of sulfation. When the volumeratio of the chlorosulfonic acid and pyridine was 1∶4, the volume ratio of the esterifying reagent and the polysaccharide was 4∶3, the esterification time was 1 h, and the esterification temperature was 60 ℃, the maximum degree of substitution of 1.613. The functional group changes of natto polysaccharides and esterified polysaccharides were compared by FTIR spectra. The results confirmed the existence of sulfuric acid polysaccharides. The antibacterial activity of natto polysaccharide and esterified polysaccharide was tested by filter paper. The results showed when the culture time was 24 h, the inhibitory effect of sulphated natto polysaccharide on Bntorobater sakazakii and Aeromonas hydrophila was higher than that of natto polysaccharide. With the increase of the culture time and concentration of polysaccharide, the inhibitory effect on Escherichia coli might be higher than that of natto polysaccharide.