In the current study, we evaluated the impact of two different doses of vitamin D supplementation (400 and 800 IU/day), administered lifelong via the diet, on T1D development and studied whether vitamin D intake and circulating concentrations of 25(OH)D3 were associated with peripheral and gut immunity (i.e., FoxP3+ Treg cells, CD39+CD73+ T cells, IL-10-secreting T cells, and T regulatory type 1 (Tr1) cells), intestinal barrier function, and gut microbiota composition in diabetes-prone NOD mice. Here, NT5E is linked to type 1 diabetes mellitus.