Development of type 1 diabetes (T1D) is preceded by invasive insulitis. Although CD4+ CD25+ regulatory T cells (nTregs) induce tolerance that inhibits insulitis and T1D, the in vivo cellular mechanisms underlying this process remain largely unclear. Using an adoptive transfer model and noninvasive imaging-guided longitudinal analyses, we found nTreg depletion did not affect systemic trafficking and tissue localization of diabetogenic CD4+ BDC2. 5 T (BDC) cells in recipient mice prior to development of T1D. In addition, neither the initial expansion/activation of BDC cells nor the number of CD11c+ or NK cells in islets and pancreatic lymph nodes were altered. Unexpectedly, our results showed nTreg depletion led to accelerated invasive insulitis dominated by CD11c+ dendritic cells (ISL-DCs), not BDC cells, which stayed in the islet periphery. Compared with control mice, the phenotype of ISL-DCs and their ability to stimulate BDC cells did not change during invasive insulitis development. However, ISL-DCs from nTreg-deficient recipient mice showed increased in vitro migration toward CCL19 and CCL21. These results demonstrated invasive insulitis dominated by DCs, not CD4+ T cells, preceded T1D onset in the absence of nTregs, and suggested a novel in vivo function of nTregs in T1D prevention by regulating local invasiveness of DCs into islets, at least partly, through regulation of DC chemotaxis toward CCL19/CCL21 produced by the islets.