Although regulatory T cells (Tregs) have been implicated in inflammatory bowel disease, Tregs from Crohn's disease (CD) patients are increased in number and function normally in vitro. To clarify this disparity, we studied Treg function in vivo using a spontaneous model of CD-like ileitis. We first administered anti-CD25-depleting antibodies to SAMP1/YitFc (SAMP) mice to assess ileitis; mesenteric lymph node cells were then transferred into SCID (severe combined immunodeficient) recipients to induce colitis. CD25 depletion increased the severity of both spontaneous ileitis and adoptively transferred colitis. Interestingly, a second transfer of CD4+ CD25+ cells from untreated AKR control mice was able to ameliorate the induced colitis, whereas CD4+ CD25+ cells from untreated SAMP mice were not, suggesting a functional abnormality in SAMP Tregs. Anti-CD25 treatment in SAMP mice also induced proliferation of CD25− Foxp3+ Tregs, which had a proinflammatory intestinal T helper type 1/T helper type 2 (Th1/Th2) effector phenotype. These studies demonstrate Treg dysfunction in a spontaneous model of CD-like ileitis.