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Exopolysaccharide produced by Leuconostoc pseudomesenteroides XG5 (XG5 EPS) is a potential prebiotic. The study objective is to assess the effect of XG5 EPS on autoimmune diabetes using non-obese diabetic (NOD) mouse strain. Our results indicated that XG5 EPS administration delayed the onset of autoimmune diabetes, reduced the level of diabetogenic cytokines IL-21 and promoted the frequency of Foxp3+ regulatory T cells in NOD mice. Moreover, XG5 EPS altered the gut microbiota composition, especially 50 mg·kg−1 XG5 EPS administration increased the ratio of Firmicutes to Bacteroidetes at the phylum level and the concentrations of total short-chain fatty acids (SCFAs) in NOD mice. Considering that the influence of viscosity on utilization of XG5 EPS, a dose dependent effect was not established. Taken together, oral administration of 50  mg·kg−1 XG5 EPS has a potential to delay the onset of autoimmune diabetes by modulating gut microbiota and its metabolites SCFAs in NOD mice.