Oral administration of porcine insulin has been shown to be effective in preventing the spontaneous occurrence of diabetes in the Non-Obese Diabetic (NOD) mouse model. In present study, we demonstrate that feeding 6-week-old female with 20 units of human insulin every 2-3 days for 30 days induces an active mechanism of suppression through the generation of regulatory T cells. Adult irradiated NOD males i.v. injected with 5 × 10⁶T cells from the spleens of diabetic female donors and the same number of T cells from the spleens of insulin-fed animals had less successful diabetes transfer than controls (4/15 vs. 8/16, P<0.001). Protection from clinical diabetes was associated with a reduction in severe insulitis (16.4 ± 3.6% vs. 52.3 ± 12.8%, P=0.023). However, more than 85% of the islets were inflamed. Feeding animals for 15 days reduced the magnitude of this protection since the number of successful transfer after 1 month was comparable (12/17 vs. 14/17) despite a significant delay in diabetes onset (P<0.001). No difference in the contribution of T cell subsets was noted by cytofluorometry in the spleens of treated animals. When T cell subsets from insulin-fed animals were co-injected with diabetogenic T cells, only purified CD4⁺ T cells were able to transfer protection since only 3/12 mice became diabetic after 36 days in comparison to 3/6 in the group co-injected with CD4⁺ T cells from PBS-fed animals, or 5/6 in the group injected with CD8⁺ T cells. In conclusion, active protection against autoimmune diabetes occurs during oral administration of human insulin in the NOD mouse. Regulatory CD4⁺ T cells probably interact with diabetogenic T cells within the islets. These results may have important clinical implications in humans.