Here we show that iNOS-deficient mice display enhanced classically activated M1 macrophage polarization without major effects on alternatively activated M2 macrophages. eNOS and nNOS mutant mice show comparable M1 macrophage polarization compared with wild-type control mice. Addition of N6-(1-iminoethyl)-L-lysine dihydrochloride, an iNOS inhibitor, significantly enhances M1 macrophage polarization while S-nitroso-N-acetylpenicillamine, a NO donor, suppresses M1 macrophage polarization. NO derived from iNOS mediates nitration of tyrosine residues in IRF5 protein, leading to the suppression of IRF5-targeted M1 macrophage signature gene activation. Computational analyses corroborate a circuit that fine-tunes the expression of IL-12 by iNOS in macrophages, potentially enabling versatile responses based on changing microenvironments. Finally, studies of an experimental model of endotoxin shock show that iNOS deficiency results in more severe inflammation with an enhanced M1 macrophage activation phenotype. These results suggest that NO derived from iNOS in activated macrophages suppresses M1 macrophage polarization.