IL-33/ST2 signal is important for the generation of forkhead box P3 (Foxp3)+ regulatory (Treg) cells, which contribute to immune homeostasis in the context of diseases. The aim of this study was to determine whether targeting IL-33/ST2 signal could establish immunological tolerance and prevent type 1 diabetes (T1D) in nonobese diabetic (NOD) mice. Female NOD mice treated with IL-33 for 4 weeks decreased the incidence and delayed the onset of autoimmune diabetes, whereas IL-33 did not revert blood glucose concentration and disease development in mice with new-onset diabetes. IL-33 reduced immune cell infiltration, increased the number of insulin-positive islet cells, as well as increased antiapoptosis molecule Bcl2 and reduced proapoptosis molecules Caspase3 at mRNA levels in the pancreas. IL-33 increased the expression of phosphorylated-Akt and phosphorylated-PI3K in the pancreas. Systemic administration of IL-33 increased the number of CD4+CD25+Foxp3+Treg cells and induced expression of Treg cell-associated molecules ST2 and GATA3 in splenic lymphocytes, and increased Foxp3, Ctla4, and Gata3 at the -mRNA level in pancreatic lymph nodes of NOD mice. IL-33 signaling stimulated activation of phosphorylation of p44/42 (Erk) and p38 MAPK, as well as CD39 in the spleen. Our results showed that IL-33 prevents disease development in prediabetic NOD mice, and highlight IL-33/ST2 as a potential therapeutic target to prevent T1D.