Metaflammation is characteristic of chronic metabolic inflammation, associated with increased risk of development of metabolic dysfunction-associated steatotic liver disease (MASLD). Palmitoylation of Myeloid differentiation factor 88 (MyD88) adaptor protein mediates biologically important signal transduction pathways in inflammatory responses. However, the molecular mechanisms underlying MyD88 palmitoylation contributes to lipid-induced metaflammation in the progression of MASLD is not completely understood. In this study, an increment of MyD88 palmitoylation was observed in the livers of high-fat diet fed mice, accompanied by increased lipid accumulation and an inflammatory response. Inhibition of MyD88 palmitoylation attenuated the inflammation and hepatic steatosis in HFD-induced mice. Mechanistically, palmitoylation of MyD88 activated NF-κB-p65 and p38 MAPK signals in a selenoprotein K (SelK)-DHHC6 palmitoyltransferase complex dependent pathway. Intervention of SelK SH3 binding domain reduced the palmitoylation level of MyD88 by inhibiting the interaction between SelK and DHHC6. Our findings suggest that MyD88 palmitoylation regulates the metabolic disorder and metaflammation through SelK/DHHC6-dependent pathway, cooperatively. Inhibition of MyD88 palmitoylation and SelK SH3 binding domain may represent a new therapeutic strategy for treatment of MASLD progression.Copyright © 2025. Published by Elsevier Inc.