Hesperetin, a flavonoid from citrus fruits, possess various pharmacological properties, including anti-inflammatory, anti-oxidative, anti-tumor potentials. However, the role and its mechanism in ulcerative colitis (UC) remains unclear. This study aimed to investigate the protective effects and mechanisms of hesperetin on dextran sodium sulfate (DSS) -induced colitis. Our results showed that hesperetin significantly relieved the symptoms of DSS -induced colitis and increased the expressions of zonula occludens-1 (ZO-1), occludin and mucin2 (MUC-2) as well as the decrease of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-18, HMGB1 and IL-6. Of note, results from immunohistochemistry (IHC) and western blotting indicated that hesperetin inhibited the expressions of receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL), the two key proteins of necroptosis pathway, and inactivated RIPK3/MLKL necroptosis signalling. Meanwhile, in the cell-coculture system between Caco-2 and RAW264.7 cells, hesperetin treatment significantly ameliorated the decrease of trans epithelial electric resistance (TEER) value while HS-173 (necroptosis inducer) could obviously influence the effect of hesperetin. In addition, hesperetin attenuated the LPS-induced increasing in 4-kDa fluorescein isothiocyanate-dextran (FD4) permeability while HS-173 could weaken the protective effect of hesperetin. Meanwhile, HS-173 reduced the changes in the expressions of phosphorylated RIPK3, phosphorylated MLKL, ZO-1, occludin and MUC-2 as well as TNF-α, IL-1β. These findings demonstrated hesperetin ameliorated DSS-induced colitis by maintaining the epithelial barrier via blocking the intestinal epithelial necroptosis.