Macrophages play an essential role in inflammation. Protein disulfide isomerase (PDI) is central to the redox system， which is closely linked with the inflammatory function of macrophages. However， the relationship between PDI and inflammation is still unknown. In this study， we tested the effects of PDI on inflammatory responses in RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS). Using CRISPR/Cas9 system， we found that PDI knockout suppressed migration， M1 polarization， and secretion of tumor necrosis factor-α (TNF-α) and interluekin-6 (IL-6). The repression of these inflammatory processes was accompanied by decreased production of reactive oxygen species (ROS). PDI ablation also inactivated the phosphorylation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and activated the phosphorylation of NF-κB inhibitor alpha (IκBα). These findings demonstrate that PDI knockout inhibits the inflammatory function of macrophages by decreasing ROS production and inactivating NF-κB pathway.