Inflammatory bowel disease (IBD) is a gastrointestinal disorder characterised by elevated colonic neutrophil extracellular traps (NETs), which are associated with disease severity. Formation of NETs is primarily driven by peptidyl arginine deaminase IV (PAD4) and other enzymes including myeloperoxidase (MPO) and neutrophil elastase. The present study evaluated the effect of MPO and PAD4 inhibition in dextran sodium sulfate (DSS)-induced colitis. Experimental colitis was induced in male C57BL/6 mice by 2% w/v DSS in drinking water ad libitum. Treatment groups received daily oral administration of MPO inhibitor (AZD3241; 30 mg/kg) and/or intraperitoneal injection of PAD4 inhibitor (GSK484; 4 mg/kg) 4 times over 9 Inhibition of PAD4 significantly diminished NET density in the colonic mucosa of mice insulted with DSS, reaching levels similar to that detected in control mice. Both inhibitors offered limited improvement in disease-activity-index, a scoring system that considers the extent of weight loss, stool consistency and rectal bleeding. Histology showed that MPO and/or PAD4 inhibition did not recover DSS-induced colon histoarchitectural damage whilst Alcian blue staining demonstrated that PAD4 failed to reduce goblet cell loss. The selected dosage of PAD4 inhibition also yielded no effect on inflammatory markers and antioxidant protein levels. These data sets suggest that other mechanisms may be involved in the pathogenesis of IBD, and the appropriate dosage of GSK484 requires thorough investigation.