Abstract IL-10 limits the magnitude of inflammatory gene expression following microbial stimuli and is essential to prevent inflammatory disease; however, the molecular basis for IL-10–mediated inhibition remains elusive. Using a genome-wide approach, we demonstrate that inhibition of transcription is the primary mechanism for IL-10–mediated suppression in LPS-stimulated macrophages and that inhibited genes can be divided into two clusters. Genes in the first cluster are inhibited only if IL-10 is included early in the course of LPS stimulation and is strongly enriched for IFN-inducible genes. Genes in the second cluster can be rapidly suppressed by IL-10 even after transcription is initiated, and this is associated with suppression of LPS-induced enhancer activation. Interestingly, the ability of IL-10 to rapidly suppress active transcription exhibits a delay following LPS stimulation. Thus, a key pathway for IL-10–mediated suppression involves rapid inhibition of enhancer function during the secondary phase of the response to LPS.