Objective <p>This study explores the molecular mechanism of HDAC5 in ferroptosis of intestinal epithelial cells in sepsis-induced acute intestinal injury.</p> Methods <p>A mouse model was established by cecal ligation and perforation (CLP) and an in vitro model of intestinal epithelial cells was induced by lipopolysaccharide (LPS). Immunohistochemistry, RT-qPCR, or Western blot determined the expressions of HDAC5, KLF4, and LncRNA MEG3 in cells. HDAC5 expression was reduced via lentivirus injection and siRNA transfection, followed by evaluation of intestinal tissue injury and cell injury, detection of Fe²⁺, ROS, GSH, and MDA levels, as well as detection of ACSL4 and SLC7A11 protein expressions. The permeability was evaluated by detecting the flux of fluorescein isothiocyanate-dextran. ChIP analyzed the enrichment of HDAC5 and H3K27ac on KLF4 promoter. The binding of KLF4 to LncRNA MEG3 promoter was verified by ChIP and dual luciferase assays. RIP and RNA pull down analyzed the binding of LncRNA MEG3 to LSD1. The enrichment of LSD1, H3K4me2, or H3K9me2 on ACSL4 or SLC7A11 promoter was analyzed by ChIP.</p> Results <p>HDAC5 expression was increased in intestinal tissues of CLP mice and LPS-induced cells, while KLF4 and LncRNA MEG3 expressions were decreased. Low expression of HDAC5 alleviated intestinal tissue injury, enhanced LPS-induced cell viability, and reduced ferroptosis. Mechanistically, HDAC5 inhibited KLF4 expression through deacetylation of H3K27ac, thereby suppressing transcriptional promotion of LncRNA MEG3 by KLF4, reducing recruitment of LSD1, enhancing H3K4me2 on ACSL4 promoter and H3K9me2 enrichment on SLC7A11 promoter, promoting ACSL4 and inhibiting SLC7A11 expression.</p> Conclusion <p>HDAC5 promotes intestinal epithelial cell ferroptosis and exacerbates sepsis-induced acute intestinal injury by inhibiting KLF4/LncRNA MEG3 axis through deacetylation of H3K27ac.</p>