Proinflammatory cytokine production in macrophages involves multiple regulatory mechanisms, which are affected by environmental and intrinsic stress. In particular, accumulating evidence has suggested epigenetic control of macrophage differentiation and function mainly in vitro. SET domain, bifurcated 1 (Setdb1, also known as Eset) is a histone 3 lysine 9 (H3K9)-specific methyltransferase and is essential for early development of embryos. Here we demonstrate that Setdb1 in macrophages potently suppresses Toll-like receptor 4 (TLR4)-mediated expression of proinflammatory cytokines including interleukin-6 through its methyltransferase activity. As a molecular mechanism, Setdb1-deficiency decreases the basal H3K9 methylation levels and augments TLR4-mediated NF-κB recruitment on the proximal promoter region of interleukin-6, thereby accelerating interleukin-6 promoter activity. Moreover, macrophage-specific Setdb1-knockout mice exhibit higher serum interleukin-6 concentrations in response to lipopolysaccharide challenge and are more susceptible to endotoxin shock than wildtype mice. This study provides evidence that the H3K9 methyltransferase Setdb1 is a novel epigenetic regulator of proinflammatory cytokine expression in macrophages in vitro and in vivo. Our data will shed insight into the better understanding of how the immune system reacts to a variety of conditions.