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Background: Cigarette smoking is emerging as an important lifestylefactor that affects intestinal function. Epidemiological studies indicated that cigarette smoking influences the course of inflammatory bowel disease and is a risk factor for colorectal cancer andintestinal polyp development. The mechanism via which cigarette smoke exerts its effect on the intestinal epithelium is unclear. These may involve indirect effects via alterations in the immune system or direct effects of cigarette smoke on the intestinal epithelium. Little is known about these direct effects of cigarette smoke on intestinal epithelial cells. Here we present an in-vitro study of these direct effects Methods: Human intestinal epithelial Caco-2 cell-matrigel-embedded spheriods were grown for 7 days in the presence of, or pre treated with, cigarette smoke extract (CSE). For pre treatment cell were incubated with CSE for 24 hours, and subsequently plated out in fresh in3D culture. Intestinal epithelial barrier function was measured witha FITC-dextran leakage assay. Cell polarity and morphogenesis were assessed with fluorescently labelled polarity markers Results: We found that CSE did not affect the polarity and barrier function of pre established lumen-forming Caco-2 spheroids.However, when exposed to CSE during the process of 3D morphogenesis,cells showed impaired growth and impaired de novo development of apical-basal cell polarity and tight junctions, resulting in defective self-assembly into single lumen-forming spheroids. This effect was dose-dependent and this phenotype could be induced with pre incubation with CSE as well. However, the administration of the glutathione precursor N-acetyl-L-cysteine (NAC) completely neutralised the inhibitory effects of CSE. The mechanisms by which NAC rescued the phenotype were independent of its capacity to induce glutathione production Conclusions: Our data demonstrate that CSE has direct and persistentinhibitory effects on intestinal epithelial cell polarity developmentand morphogenesis, and provide insight into the cell biologicalmechanisms. NAC supplementation was identified as a protectivestrategy for smoke-induced epithelial dysfunction