Macrophages are one of the most abundant leucocytes in the intestinal mucosa where they are essential for maintaining homeostasis. However they are also implicated in the pathogenesis of disorders such as inflammatory bowel disease (IBD), offering potential targets for novel therapies. Tissue macrophages are a heterogeneous population of immune cells that fulfill tissue-specific and niche-specific functions. These unique phenotypes likely reflect the heterogeneity of tissue macrophage origins and influence the tissue environment in which they reside. Here we describe how we can characterize and isolate the colonic macrophages.