Summary: Inflammatory bowel disease is characterized by dysregulated immune responses in inflamed intestine, with dominance of interleukin‐17 (IL‐17) ‐producing cells and deficiency of regulatory T (Treg) cells. The aim of this study was to investigate the effect and mechanisms of sirolimus, an inhibitor of the mammalian target of rapamycin, on immune responses in a murine model of Crohn's disease. Murine colitis was induced by intrarectal administration of 2,4,6‐trinitrobenzene sulphonic acid at day 0. Mice were then treated intraperitoneally with sirolimus daily for 3 days. The gross and histological appearances of the colon and the numbers, phenotype and cytokine production of lymphocytes were compared with these characteristics in a control group. Sirolimus treatment significantly decreased all macroscopic, microscopic and histopathological parameters of colitis that were analysed. The therapeutic effects of sirolimus were associated with a down‐regulation of pro‐inflammatory cytokines tumour necrosis factor‐α, IL‐6 and IL‐17A. Intriguingly, sirolimus administration resulted in a prominent up‐regulation of the regulatory cytokine transforming growth factor‐β. Supporting the hypothesis that sirolimus directly affects the functional activity of CD4⁺ CD25⁺ Treg cells, we observed a remarkable enhancement of FoxP3 expression in colon tissues and isolated CD4⁺ T cells of sirolimus‐treated mice. Simultaneously, sirolimus treatment led to a significant reduction in the number of CD4⁺ IL‐17A⁺ T cells in the mesenteric lymph node cells as well as IL‐17A production in mesenteric lymph node cells. Therefore, sirolimus may offer a promising new therapeutic strategy for the treatment of inflammatory bowel disease.