process of intestinal inflammation is less understood. While we could demonstrate that HDAC inhibitors induce amelioration of colitis in mice, we are further interested in how far specific HDAC are responsible for inflammatory responses and what are the mechanism behind these effects. Methods: The macrophage cell line RAW 264.7 and the monocyte cell line U937 were analysed for HDAC5-expression using quantitative PCR and Western-blot. The cells were transfected with a HDAC5-encoding plasmid for overexpression or small interfering (si)RNA for knock-down studies. HDAC5 knock out mice were used in dextran sulphate sodium (DSS) induced colitis model. Colon tissue was analysed by histology and lamina propria mononuclear cells (LPMCs) were isolated. All cells were analysed by flow cytometry. Supernatant of colon and cell cultures were analysed by cytometric bead assay. Results: In RAW 264.7 and U937 expression of HDAC5 mRNA down regulated for up to 7 h but presented baseline levels after 24 h. HDAC5-overexpression in RAW 264.7 resulted in an increased LPS-dependent secretion of the pro-inflammatory cytokines tumor necrosis factor alpha, the monocyte chemoattractant protein-1 and interleukin 6. Subsequently, knock-down of HDAC5-mRNA expression by specific siRNA significantly reduced the secretion of these cytokines. These effects were accompanied by increased nuclear factor kappa B activity. Further experimental colitis experiments demonstrate a differential disease pattern for HDAC5 knockout mice compared to wild type mice. This was shown by an increased histological disease score, higher weight loss and severe intestinal bleeding. LPMC isolation followed by extensive FACS analysis of the LPMC confirmed these results by demonstrating changes in macrophage polarization. When the colonic barrier function was analysed in DSS induced colitis experiments, we detected a decreased trans-epithelial resistance and modulated flux of mannitol, HRP and FITC-Dextran. Conclusion: Our data demonstrate a critical function of HDAC5 in the pro-inflammatory immune response of macrophages in the lamina propria as well as a role within the physiological barrier of the gut epithelium, proposing a potential new target for the treatment of inflammatory bowel disease