A role for dipeptidyl peptidase IV (DPP IV/CD26) in the pathogenesis of inflammatory bowel disease has been proposed owing to its involvement in immune regulation via its expression on immune cells and ability to cleave biologically active molecules. The aim of this study was to investigate the influence of DPP IV/CD26 deficiency on development and resolution of dextran sulfate sodium‐induced colitis in CD26‐deficient (CD26^‐/‐) and wild‐type (C57BL/6) mice. Colitis was characterized by clinical and histological changes and infiltration of immune cells in the colonic mucosa. In the acute phase of colitis, loss of body mass and disease activity in C57BL/6 mice was more intensive than in CD26^‐/‐ mice, in spite of similar histopathological changes at the local level. Although acute inflammation induced a significant increase in the number of macrophages and dendritic cells in both mouse strains, in CD26^‐/‐ mice the increase of macrophages was twice that in C57BL/6 animals (18.0 ± 4.5 versus 41.3 ± 5.8), whereas the increase in dendritic cells was more pronounced in C57BL/6 mice. In the acute phase of colitis, colonic DPP IV/CD26 activity was significantly decreased in C57BL/6 mice compared with healthy animals. The results of our study reveal that DPP IV/CD26 deficiency affects the onset of clinical symptoms and the specific cells infiltrating at the site of inflammation in CD26^‐/‐ animals, suggesting a pathophysiological role of DPP IV/CD26 and providing new insights into the nature of the immune response activated during the development of colitis.