Abstract: In order to understand the mechanism of uracil on the growth by Aureobasidium pullulans and synthesis of pullulan, the optimal dosage and time of uracil in pullulan polysaccharide fermentation were studied. The label-free quantitative method and liquid chromatography-tandem mass spectrometry were used to compare the protein components in the late fermentation stage (88 h) by Aureobasidium pullulans and the differential proteins were analyzed by bioinformatics. The results showed that adding 0.5g/L uracil at 48 h had the most significant increasing in pullulan polysaccharide yield, which was verified on 5 L fermenter from 70.13 to 86.27 g/L, increasing by 23%. Protein component analysis identified 80 different proteins, including 40 increases protein and 40 lower protein (difference>2, P<0.05), the protein of the differences of clustering analysis, GO function enrichment, KEGG pathway analysis showed that the differences in protein involved in cellular processes, metabolic processes, and other important biological processes. The differential proteins were mainly involved in glycolysis, metabolism of fructose and mannose, metabolism of glyoxylic acid and dicarboxylic acid, pyruvate metabolism and TCA cycle, which ultimately led to the change of pullulan polysaccharide yield. These results would provide a molecular basis for further understanding the metabolic mechanism of pullulan polysaccharides produced by Aureobasidium pullulans.