Abnormalities of dendritic cells (DCs) have been identified in type 1 diabetic patients and in nonobese diabetic (NOD) mice that are associated with augmented nuclear transcription factor (NF)-κB activity. An imbalance that favors development of the immunogenic DCs may predispose to the disease, and restoration of the balance by administration of DCs deficient in NF-κB activity may prevent diabetes. DCs propagated from NOD mouse bone marrow and treated with NF-κB-specific oligodeoxyribonucleotide (ODN) in vitro (NF-κB ODN DC) were assessed for efficacy in prevention of diabetes development in vivo. Gel shift assay with DC nuclear extracts confirmed specific inhibition of NF-κB DNA binding by NF-κB ODN. The costimulatory molecule expression, interleukin (IL)-12 production, and immunostimulatory capacity in presenting allo- and islet-associated antigens by NF-κB ODN DC were significantly suppressed. NF-κB ODN renders DCs resistant to lipopolysaccharide stimulation. Administration of 2 × 106 NF-κB ODN DCs into NOD mice aged 6-7 weeks effectively prevented the onset of diabetes. T-cells from pancreatic lymph nodes of NF-KB ODN DC-treated animals exhibited hyporesponsiveness to islet antigens with low production of interferon-γ and IL-2. These findings provide novel insights into the mechanisms of autoimmune diabetes and may lead to development of novel preventive strategies.