Resident intestinal bacteria likely play an important role in the pathogenesis of Crohn’s disease through their interaction with the gut immune system. SAMP1/YitFc mice spontaneously develop chronic, discontinuous, transmural ileitis with many features similar to Crohn’s disease. The aim of this study was to determine the effects and elucidate the mechanisms of action of antibiotic treatment in the SAMP1/YitFc mouse model of ileitis. Mice were treated orally with ciprofloxacin and metronidazole before the development of ileitis (prevention protocol) or after ileitis was fully established (treatment protocol). Terminal ilea were harvested for histological scoring, and lamina propria and mesenteric lymph node cells were isolated for analysis of activation markers and cytokine production. Antibiotic therapy significantly decreased the severity of ileitis both in the prevention (40% reduction, p< 0.05) and the treatment (25% reduction, p< 0.01) protocols, compared with untreated, control mice. These effects were associated with a decreased percentage of CD4+/CD45RB high lymphocytes in mesenteric lymph nodes of antibiotic-treated mice, as well as decreased production of IFN-γ (prevention: 0.53±0.21 vs 1.84±0.04 ng/ml, p< 0.05; treatment: 8.4±0.4 vs 12.4±0.7 ng/ml, p< 0.005) and TNF (prevention: 61.5±13 vs 134±19 pg/ml, p< 0.01; treatment: 333.5±11 vs 496±20 pg/ml, p< 0.001). The number of activated lamina propria lymphocytes was also reduced after antibiotic treatment. In conclusion, antibiotic therapy significantly ameliorates the severity of ileitis in SAMP1/YitFc mice by a mechanism involving down-regulation of activated gut lymphocytes and inhibition of intestinal Th1 cytokine production.