Forkhead box 'Other' (FoxO) proteins, a subgroup of the Forkhead transcription factor family, play an important role in mediating the effects of insulin and growth factors on diverse physiological functions. In this study, we investigated the role of FoxO4 in the relationship between alcohol liver disease and intestinal barrier dysfunction using an animal model. Six to eight-week-old male WT rats were divided into eight groups. They were separately administered corn starch dissolved in PBS; 40% alcohol (5 g/kg body weight) through stomach feeding every 12 h/time, three times in total; tumor necrosis factor α (TNFα) (10 µg/kg) injected intraperitoneally 30 min before alcohol administration; wortmannin (1.4 mg/kg) 30 min before alcohol administration; IGF-1 (0.2 mg/kg) 30 min before alcohol administration; anti-TNFα (5 mg/kg) injected intravenously 30 min before alcohol administration. In addition, two placebo groups were treated with PBS either intraperitoneally or intravenously prior to alcohol administration. TNFα and endotoxin in plasma were measured by ELISA and Tachypleus Amebocye Lysate assays. Immunohistochemistry and western blotting were used to identify the mechanisms of FoxO4 action in regulating epithelial permeability. Furthermore, electron microscopy, reverse transcription-polymerase chain reaction and western blotting were used to examine the expression of tight junction proteins and nuclear factor-κB (NF-κB). Compared with the control group, TNFα in the alcohol group was significantly higher. TNFα could induce FoxO4 phosphorylation; p-FoxO4 was limited into the cytoplasm and inactivated; inactive FoxO4 which was in high levels lost the ability to suppress NF-κB. Therefore, the expression of NF-κB was increased and it downregulated tight junction protein (including ZO-1 and occludin) expression, and increased epithelial permeability. As a result, intestinal bacteria grew excessively, endotoxin was released into the portal circulation and liver injury deteriorated. These results indicate that a complex network of mechanisms is involved in the beneficial effects of FoxO4 in epithelial barrier dysfunction. TNFα can upregulate phosphorylation of FoxO4. FoxO4 which is located in the nucleus is limited into the cytoplasm and inactivated; it loses the ability to suppress NF-κB activity, it downregulates the expression of tight junction proteins and increases epithelial permeability. Disruption of the intestinal barrier allows endotoxin and other bacterial products in the gut lumen to pass into the portal circulation and cause hepatic inflammation. At the same time, the changes of liver injury deteriorate.