This study investigates the role of the H3K27me3-specific demethylase subfamily (KDM6 subfamily members JMJD3 and UTX) in regulating proinflammatory macrophage responses. The researchers used a structure-guided approach to design a small-molecule inhibitor, GSK-J1, which selectively targets these enzymes. High-resolution crystal structures of human and mouse JMJD3 were solved, revealing the binding mode of the H3K27me3 peptide and the inhibitor. GSK-J1 binds to the catalytic site of JMJD3, mimicking the cofactor α-ketoglutarate and the substrate peptide, and inhibits H3K27 demethylation. In human primary macrophages, GSK-J1 reduces lipopolysaccharide-induced proinflammatory cytokine production, specifically targeting JMJD3 and UTX. The study provides insights into the functional role of H3K27 demethylases in inflammatory responses and suggests potential therapeutic applications for selective inhibitors of the KDM6 subfamily.This study investigates the role of the H3K27me3-specific demethylase subfamily (KDM6 subfamily members JMJD3 and UTX) in regulating proinflammatory macrophage responses. The researchers used a structure-guided approach to design a small-molecule inhibitor, GSK-J1, which selectively targets these enzymes. High-resolution crystal structures of human and mouse JMJD3 were solved, revealing the binding mode of the H3K27me3 peptide and the inhibitor. GSK-J1 binds to the catalytic site of JMJD3, mimicking the cofactor α-ketoglutarate and the substrate peptide, and inhibits H3K27 demethylation. In human primary macrophages, GSK-J1 reduces lipopolysaccharide-induced proinflammatory cytokine production, specifically targeting JMJD3 and UTX. The study provides insights into the functional role of H3K27 demethylases in inflammatory responses and suggests potential therapeutic applications for selective inhibitors of the KDM6 subfamily.