Objective Increased neutrophil extracellular trap-osis (NETosis) is a hallmark of inflammatory bowel disease (IBD). However, the mechanisms of NETosis formation and its detrimental effect on the intestinal epithelium remain poorly understood. Design Dextran sulfate sodium (DSS) induce colitis in peptidyl-arginine deiminase-4 deficient (PAD4-/-) and wild-type (WT) mice. PAD4, NETosis and citrullination levels were detected in mice and human volunteers. Citrullination analysis and single-cell RNA-sequencing (scRNA-seq) were performed to explore the target of PAD4 during NETosis formation. The effect of this citrullinated target in the intestinal epithelium during the pathogenesis of IBD was validated both in vivo and in vitro. Results The levels of NETosis and citrullination were improved in IBD patients, which was positively associated with endoscopic inflammation scores. Deleting PAD4 alleviated colonic inflammation and intestinal barrier dysfunctions. In citrullination analysis, R243 of mitochondrial creatine kinase 1 (CKMT1) was identified as the potential target of PAD4 during NETosis. The scRNA-seq data further indicated that PAD4 in neutrophils contributed to the increase of epithelia (Nos2+ Clca3b+) and the decrease of distal immature epithelia. PAD4-mediated NETosis contributed to the destruction of intestinal barriers and increase of apoptosis as well as lower level of CKMT1 protein in vitro. Finally, mice with intestinal epithelial-specific ablation of CKMT1 were used to verify its detrimental role in the development of IBD, which was also validated by the moderate correlation between CKMT1 and endoscopic scores in IBD patients.