Abstract IL-10 is a critical regulator of colitis development. Yet, its cellular targets and mechanisms of action are not fully resolved. To identify lineages and mechanisms responsible for IL-10's protective effects in colitis, we generated mice with cell type-restricted deficiencies in IL-10 responsiveness by using DSS colitis model with mice bearing deletions of the IL-10Rα in T cells, B cells, DCs, or macrophages. Mice with a macrophage-selective deletion of IL-10Rα (IL-10RαMdel) developed markedly enhanced 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-10RαMdel colitis was associated with increased mucosal barrier disruption in the setting of intact epithelial regeneration. Quantitation and phenotypic assessment of lamina propria macrophages did not identify differences from controls. IL-10Rα-deficient macrophages also did not display a competitive advantage over wild type cells in mixed chimeras. Pro-inflammatory cytokine production, and particularly TNF-α, was increased, though TNF-α neutralization failed to reveal a defining role for the cytokine in the aggravated disease. IL-10RαMdel lamina propria macrophages produced substantially greater levels of NO and ROS than control macrophages. Inhibition of these more dramatically reduced colitis severity in IL-10RαMdel mice, and largely eliminated the differential effect of DSS in IL-10RαMdel and control mice.