Inflammatory bowel disease reflects an aberrant mucosal CD4+ T cell response to commensal enteric bacteria. In addition to regulatory T cell subsets, recent studies have revealed a protective role of B cells in murine CD4+ T cell colitis, but the relationship of their action to T cell immunoregulation is unknown. Here we report that mesenteric lymph node (MLN) B cells protect mice from colitis induced by Galphai2-/- CD4+ T cells. Protection required the transfer of both B cells and CD8alpha+ T cells; neither cell type alone was sufficient to inhibit CD4+ T cell-mediated colitis. Similar results were also observed in colitis induced by CD4+CD45RBhi T cells. Immunoregulation was associated with localization of B cells and expansion of CD4-CD8- CD3+NK1.1+ T cells in the secondary lymphoid compartment, as well as expansion of CD4+CD8alpha+ T cells in the intestinal intraepithelial compartment. MLN B cells from Galphai2-/- mice were deficient in a phenotypic subset and failed to provide cotransfer colitis protection. These findings indicate that protective action of B cells is a selective trait of MLN B cells acquired through a Galphai2-dependent developmental process and link B cells with the formation of regulatory T cells associated with mucosal immune homeostasis.