NOD-like receptor protein 3 (NLRP3) inflammasome activation triggers inflammation progression in some metabolism disorders, frequently accompanying the up-regulation of galectin-3 (Gal-3). However, the precise mechanisms of Gal-3 activating NLRP3 inflammasome remain unclear in nonalcoholic steatohepatitis (NASH).Male C57BL/6J were fed by high-fat diet (HFD) for 32 weeks to induce NASH and then the hepatic damage, cytokines, Gal-3 and TLR4 expression, and NLRP3 inflammasome activation were examined. Such indicators were similarly determined when HepG2 cells were co-incubated with palmitic acid (PA, 200 μM), β-lactose, and TAK-242, or pre-transfected with TLR4. Immunofluorescence, immunohistochemistry, and co-immunoprecipitation were conducted to confirm the potential interaction between Gal-3 and TLR4. To further identify the inflammatory regulation roles of Gal-3 and its terminals in TLR4/NLRP3, HepG2 cells were transfected with Gal-3 and its variants.Chronic HFD induced sustained hepatic steatosis and inflammatory injury, with increased inflammatory cytokines, Gal-3 and TLR4 expression, and NLRP3 inflammasome activation. Similar changes were found in PA-dosed HepG2 cells, which were rescued by β-lactose but deteriorated with TLR4 overexpression. However, TAK-242 treatment decreased AST, ALT, cytokines, and normalized NLRP3, caspase-1, and ASC expression. Furthermore, TLR4 was pulled down when Gal-3 was enriched. Only full-length Gal-3 and its carbohydrate recognition domain (CRD) promoted cytokines, TLR4 expression, and NLRP3 inflammasome activation.Gal-3 may induce chronic HFD-derived NASH progression by activating TLR4-mediating NLRP3 inflammasome via its CRD, which sheds new light on candidate target for the treatment and prevention of NASH inflammation despite further research for its precise roles in the future.Copyright © 2022. Published by Elsevier Inc.