Breakdown of the epithelial barrier because of toxins or other insults leads to severe colitis. Interleukin-10 (IL-10) is a critical regulator of this, yet its cellular targets and mechanisms of action are not resolved. We address this here. Mice with a macrophage-selective deletion of IL-10Rα (IL-10Rα(Mdel)) developed markedly enhanced dextran sodium sulfate (DSS)-induced colitis that did not significantly differ from disease in IL-10(-/-) or IL-10Rα(-/-) mice; no impact of IL-10Rα deficiency in other lineages was observed. IL-10Rα(Mdel) colitis was associated with increased mucosal barrier disruption in the setting of intact epithelial regeneration. Lamina propria macrophages (LPMφs) did not show numerical or phenotypic differences from controls, or a competitive advantage over wild-type cells. Proinflammatory cytokine production, and particularly tumor necrosis factor-α (TNF-α), was increased, although TNF-α neutralization failed to reveal a defining role for this cytokine in the aggravated disease. Rather, IL-10Rα(Mdel) LPMφs produced substantially greater levels of nitric oxide (NO) and reactive oxygen species (ROS) than controls. Inhibition of these had modest effects in wild-type mice, although they dramatically reduced colitis severity in IL-10Rα(Mdel) mice, and largely eliminated the differential effect of DSS in them. Therefore, the palliative actions of IL-10 in DSS-induced colitis predominantly results from its macrophage-specific effects. Downregulation of NO and ROS production are central to the protective actions of IL-10.