Background: Intestinal macrophages play an important role in mucosal inflammation. In normal colonic mucosa we recently demonstrated a unique macrophage phenotype with attenuated immune functions. Here we present an analysis of the alterations of the phenotype of colonic macrophages in inflammatory bowel disease (IBD). Methods: Intestinal macrophages were isolated from biopsies of patients with IBD (n = 20). Flow cytometric triple fluorescence analysis was applied to study CD14, CD16, CD33, HLA‐DR, CD44, CD11b, CD11c and CD3/CD19 expression. Results: In IBD there was an increase in expression not only of CD14 compared to control mucosa (36.0%±13.2% vs. 10.5%±3.8%, P<0.0001) but also of CD16 (28.6%±10.3% vs. 10.1%±3.9%, P<0.0001), HLA‐DR (53.1%±15.9% vs. 27.3%± 9.2%, P<0.0005), CD11b (42.8%±14.2% vs. 17.4%±6.8%, P<0.0001) and CD11c (35.1% ± 15.9% vs. 17.8%±10.4%, P<0.005.). Furthermore, a hitherto undescribed new population of macrophages could be detected by flow cytometry only in patients with ulcerative colitis (CD16++, CD11b++, CD14low, CD33low, CD11c‐) accounting for 5.8% of all cells isolated. Conclusion: In contrast to colonic macrophages from normal mucosa, there is a significantly higher expression of CD14, CD16, HLA‐DR, CD11b and CD11c in IBD, indicating additional macrophage populations in the inflamed mucosa. This may reflect either a recruitment of new cells from the circulation or a change in phenotype of resident cells.