Gut ischemia and hypoxia post severe burn leads to breakdown of intestinal epithelial barrier and enteric bacterial translocation (EBT), resulting in serious complications, such as systemic inflammatory response syndrome, sepsis and multiple organ failure. Cystic fibrosis transmembrane conductance regulator (CFTR) is known to be downregulated by hypoxia and modulate junctional complexes, which are crucial structures maintaining the intestinal barrier. This study aimed to investigate whether CFTR plays a role in both regulating the intestinal barrier and mediating EBT post severe burn, as well as the signaling pathways involved in these processes. An in vitro Caco-2 cell model subjected to hypoxic injury and an in vivo mouse model with a 30% total body surface area full-thickness dermal burn were established. DF 508 mice (mice with F508del CFTR gene mutation) were used as an in vivo model to further demonstrate the role of CFTR in maintaining normal intestinal barrier function. QRT-PCR, western blot, ELISA, TER assay and immunofluorescence staining were used to detect the expression and localization of CFTR and tight junction proteins, as well as the function of tight junctions. Our data indicated that, in Caco-2 cells, the hypoxia condition significantly reduced CFTR expression; activated extracellular signal-regulated kinase and nuclear factor-κB signaling; elevated secretion of inflammatory factors (tumor necrosis factor-α, interleukin-1β and interleukin-8); downregulated zonula occludens-1, occludin and E-cadherin expression; decreased transepithelial electrical resistance values; and led to a cellular mislocation of ZO-1. More importantly, knockdown of CFTR caused similar alterations. The upregulation of inflammatory factors and downregulation of tight junction proteins (ZO-1 and occludin) induced by knockdown of CFTR could be reversed by specific extracellular signal-regulated kinase or nuclear factor-κB inhibition. In support of the in vitro data, exuberant secretion of pro-inflammatory mediators and EBT was observed in the intestine of severely burnt mice in vivo. EBT occurred in DF508 mice (mice with the F508del CFTR gene mutation), accompanied by augmented tumor necrosis factor-α, interleukin-1β and interleukin-8 levels in the ileum compared to wildtype mice. In addition, vitamin D3 was shown to protect the intestinal epithelial barrier from hypoxic injury. Collectively, the present study illustrated that CFTR and downstream signaling were critical in modulating the intestinal epithelial junction and EBT post severe burn.