Cl-amidine, a peptidylarginine deiminase inhibitor, has been shown to ameliorate the disease course and clinical manifestation in variety of disease models. Due to the beneficial effects of Cl-amidine, it has been becoming the hottest compound for the study in inflammatory diseases. However, the anti-inflammatory activity of Cl-amidine in lipopolysaccharide (LPS)-induced mouse mastitis remains unclear. In this study, we investigated the effects of Cl-amidine on LPS-induced mastitis mouse model. The mouse mastitis model was established by injection of LPS through the canals of the mammary gland. Cl-amidine was administered intraperitoneally 1 h before LPS treatment. The results showed that Cl-amidine significantly attenuated the damage of the mammary gland, which suppressed the activity of myeloperoxidase (MPO). The real-time PCR results indicated that Cl-amidine inhibited the production of TNF-α, IL-1β and IL-6 in LPS-induced mouse mastitis. Moreover, the western blot results indicated that Cl-amidine decreased the phosphorylation of IκB, p65, p38, ERK and the expression of NLRP3 in LPS-induced mouse mastitis. Furthermore, the neutrophils extracellular traps (NETs) were determined by Quant-iT picogreen dsDNA assay kit®, which suggested that Cl-amidine significantly inhibited the NETs in mouse serum. This study demonstrated that Cl-amidine decreased the pathological injury in LPS-induced mouse mastitis by inhibiting NF-κB, MAPK, NLRP3 signaling pathway and NETs release, which provides a potential candidate for the treatment of mastitis. •Cl-amidine reduces inflammation in vivo.•Cl-amidine attenuates LPS-induced mouse mastitis.•Cl-amidine inhibits the production of TNF-α, IL-1β and IL-6 in LPS-induced mouse mastitis.