Oral tolerance, induced by oral administration of autoantigens, is a promising therapeutic approach to treat type 1 diabetes mellitus (T1DM). However, the degradation of antigens passing through the gastrointestinal tract (GIT) leads to low induction efficiency. Based on our previous study, a single-chain insulin (SCI-59) analog, bound to the surface of lactic acid bacteria (LAB) bacterium-like particles (BLPs), was more stable in the simulated gastric fluid, compared to free SCI-59 and insulin. Based on the analysis of diabetes progression, a significant decrease in the incidence of diabetes was observed in mice fed BLPs-SCI-59. Oral administration of BLPs-SCI-59 can enhance glucose tolerance in NOD mice and this effect may result from the protection of pancreatic islet beta cells, as compared to the free SCI-59 group and BLPs group. Oral administration of BLPs-SCI-59 can significantly reduce insulitis and preserve the ability of insulin secretion in treated mice. Oral vaccination with BLPs-SCI-59 induced SCI-59 specific T cell tolerance in treated mice, which may due to the repair of Th1/Th2 imbalance and increased CD4CD25FoxP3 regulatory T cells (Tregs). These results show that oral vaccination with BLPs-SCI-59 is a promising way to prevent T1DM in NOD mice.