Neutrophils release neutrophil extracellular traps (NETs), which are extracellular chromatin decorated with histones and antimicrobial proteins. Although known for antimicrobial properties, overzealous production of NETs (NETosis) may lead to cytotoxicity and multiple organ failure in sepsis. Pathogen-induced NETosis has been extensively studied in mice but its importance in dogs remains largely unknown. This study sought to characterize in vitro NETosis induced by E.coli LPS, including assessing the role of peptidylarginine deiminase (PAD) in canine NETosis. Neutrophils (1×10 cells/ml) from healthy dogs were isolated and treated with 100μg/ml LPS, 100nM phorbol 12-myristate 13-acetate (PMA), or buffer for either 90 or 180min. NETs were assessed using fluorescence microscopy of living neutrophils and immunofluorescent microscopy. Supernatant and cellular debris were purified and cell-free DNA was quantified by spectrophotometry. The role of PAD was assessed by treating LPS- and PMA-activated neutrophils with 50, 100 or 200μM of the PAD inhibitor, Cl-amidine. In vitro NETosis was characterized by co-localization of cell-free DNA, citrullinated histone H3, and myeloperoxidase. LPS stimulation resulted in intracellular citrullination of histone H3. Compared to PMA chemically-induced NETosis, LPS resulted in smaller NETs with less extracellular citrullinated histone H3. Cl-amidine decreased citrullination of histones and NET production in either LPS- or PMA-stimulated neutrophils demonstrating that neutrophil PAD is essential for these cellular processes.