Several subsets of innate immune cells, all with unique properties, reside within the intestinal lamina propria. However, compared with intestinal dendritic cells (DCs), intestinal macrophages are less well characterized. In this study, we examined the properties of macrophages in the colonic lamina propria (LMφ). Colonic DCs (LDC) showed LPS-induced production of IL-12p40. In contrast, LMφ showed constitutive IL-10 production and unresponsiveness to LPS in terms of inflammatory cytokine production. Comparison of the gene expression profiles between LMφ and LDC revealed that LMφ preferentially expressed IL-10-related genes. LMφ obtained from mice lacking IL-10 or Stat3 showed hyperproduction of tumour necrosis factor (TNF)-α and IL-6 in response to LPS. IL-10 production in the large intestine was mainly induced by LMφ and regulatory T cells and was dependent on the presence of commensal microbiota. Accordingly, LMφ from germ-free mice showed less production of IL-10 and increased levels of LPS-induced TNF-α and IL-6 production. Taken together, these results demonstrate that the activity of LMφ to produce pro-inflammatory cytokines is negatively regulated through commensal microbiota-dependent IL-10 production in the large intestine.