Metabolic dysfunction-associated steatohepatitis (MASH)-fibrosis is a reversible stage of liver disease accompanied by inflammatory cells infiltration. Neutrophils extrude a meshwork of chromatin fibers to establish neutrophil extracellular traps (NETs), which play important roles in inflammatory response regulation. Our previous work demonstrated that NETs promote hepatocellular carcinoma in MASH. However, it is still unknown if NETs play a role in the molecular mechanisms of liver fibrosis.Following 12 weeks of Western diet (WD)/carbon tetrachloride (CCl4), MASH-fibrosis was identified in C57BL/6 mice with increased NET formation. However, NET depletion using DNase I treatment or mice knocked-out for peptidyl arginine deaminase type IV (PAD4-/-), significantly attenuated the development of MASH-fibrosis. NETs were demonstrated to induce hepatic stellate cells (HSCs) activation, proliferation, and migration through augmented mitochondrial and aerobic glycolysis to provide additional bioenergetic and biosynthetic supplies. Metabolomic analysis revealed markedly an altered metabolic profile upon NET stimulation of HSCs that were dependent on arachidonic acid metabolism. Mechanistically, NET stimulation of toll-like receptor 3 (TLR3) induced cyclooxygenase-2 (COX-2) activation and prostaglandin E2 (PGE2) production with subsequent HSC activation and liver fibrosis. Inhibiting COX-2 with celecoxib reduced fibrosis in our MASH model.Our findings implicate NETs playing a critical role in the development of MASH-hepatic fibrosis by inducing metabolic reprogramming of HSCs via the TLR3/COX-2/PGE2 pathway. Therefore, NET inhibition may represent an attractive treatment target for MASH liver fibrosis.Copyright © 2024 American Association for the Study of Liver Diseases.