The determinant spreading of T cell autoimmunity plays an important role in the pathogenesis of type 1 diabetes and in the protective mechanism of Ag-based immunotherapy in NOD mice. However, little is known about the role of APCs, particularly B cells, in the spreading of T cell autoimmunity. We studied determinant spreading in NOD/scid or Igμ−/− NOD mice reconstituted with NOD T and/or B cells and found that mice with mature B cells (TB NOD/scid and BMB Igμ−/− NOD), but not mice that lacked mature B cells (T NOD/scid and BM Igμ−/− NOD), spontaneously developed Th1 autoimmunity, which spread sequentially among different β cell Ags. Immunization of T NOD/scid and BM Igμ−/− NOD mice with a β cell Ag could prime Ag-specific Th1 or Th2 responses, but those T cell responses did not spread to other β cell Ags. In contrast, immunization of TB NOD/scid and BMB Igμ−/− NOD mice with a β cell Ag in IFA induced Th2 responses, which spread to other β cell Ags. Furthermore, we found that while macrophages and dendritic cells could evoke memory and effector T cell responses in vitro, B cells significantly enhanced the detection of spontaneously primed and induced Th1 responses to β cell Ags. Our data suggest that B cells, but not other APCs, mediate the spreading of T cell responses during the type 1 diabetes process and following Ag-based immunotherapy. Conceivably, the modulation of the capacity of B cells to present Ag may provide new interventions for enhancing Ag-based immunotherapy and controlling autoimmune diseases.