Peptidylarginine deiminase 2 (PAD2), an enzyme that converts peptidyl-arginine to peptidyl-citrulline, is widely distributed in various tissues and cells. Although PAD4 is known to be involved in various inflammatory and immune diseases via neutrophil extracellular trap (NETosis) formation, the pathophysiological roles of PAD2 have not been fully understood. The present study investigated the pathogenic role of PAD2 in inflammatory bowel disease using trinitrobenzene sulfonic acid (TNBS)-induced murine colitis model. PAD2-deficent (PAD2KO) mice were generated by CRISPR-Cas9-mediated genomic editing. Colitis was induced in PAD2KO and its wild-type (WT) mice by intrarectal injection of TNBS. The extracellular trap formation was detected by triple immunohistochemical staining with myeloperoxidase (MPO), citrullinated histone H3, and DNA (Sytox Blue). TNBS injection induced body weight loss, extensive colonic erosions, and ulceration accompanied by an increase in MPO activity in WT mice, but these responses were significantly attenuated in KO mice. Daily administration of Cl-amidine (an inhibitor of pan-PADs) or DNase Ⅰ (an inhibitor of extracellular trap formation) also significantly reduced the severity of TNBS-induced colitis. Furthermore, the extracellular trap formation was upregulated by TNBS injection at lesion sites, but this response was apparently attenuated by PAD2KO and Cl-amidine injection. These findings suggest that PAD2 contributes to the pathogenesis of TNBS-induced colitis probably via inhibition of extracellular traps.