The current study aimed to establish a non-alcoholic fatty liver disease (NAFLD) model using HFD-fed SD rats and FFA-stimulated human THP-1 cells to examine whether hesperidin (HSP) plays a role in endoplasmic reticulum stress (ERS)-induced inflammation in the pathogenesis of NAFLD.Oil red O staining was used to determine the effect of HSP on hepatic steatosis in rat liver tissues. Differentially expressed genes (DEGs) were subjected to functional enrichment analysis by bioinformatics. Western blotting was used to detect the protein expression of GRP94, ATF6, ATF4, p-PERK, p-IRE1α, IL-1β, IL-6, and TNF-α in liver tissues and THP-1 cell lines, and the expression of GRP94 and p-PERK in vitro was detected through immunofluorescence staining.HSP significantly decreased the weight gain, hepatic steatosis but not serum lipid profile and suppressed the serum levels of inflammatory factors in HFD-fed rats. It was revealed by bioinformatics analysis that the inflammatory response and IRE1α activation were enriched signaling pathways in NAFLD. The expression of ERS-related biomarkers, GRP94, ATF6, ATF4, p-PERK and p- IRE1α, was significantly suppressed by HSP in vivo and in vitro. Moreover, the inflammatory markers, including IL-1β, IL-6, and TNF-α, were also decreased by HSP in vivo and in vitro. Immunofluorescence staining exposed that the expression of GRP94 and p-PERK was decreased by HSP in vitro.HSP may suppress ERS-induced inflammation in the pathogenesis of NAFLD.