A critical cis-regulatory element for the cystic fibrosis transmembrane conductance regulator (CFTR) gene is located in intron 11, 100 kb distal to the promoter, with which it interacts. This sequence contains an intestine-selective enhancer and associates with enhancer signature proteins, such as p300, in addition to tissue-specific transcription factors (TFs). Here we identify critical TFs that are recruited to this element and demonstrate their importance in regulating CFTR expression. In vitro DNase I footprinting and electrophoretic mobility shift assays (EMSA) identified four cell-type selective regions that bound TFs in vitro. Chromatin immunoprecipitation (ChIP) identified forkhead box A1/A2 (FOXA1/A2), hepatocyte nuclear factor 1 (HNF1), and caudal type homeobox 2 (CDX2) as in vivo trans-interacting factors. Mutation of their binding sites in the intron 11 core compromised its enhancer activity when measured by reporter gene assay. Moreover, siRNA-mediated knockdown of CDX2 caused a significant reduction in endogenous CFTR transcription in intestinal cells, suggesting that this factor is critical for the maintenance of high levels of CFTR expression in these cells. The ChIP data also demonstrate that these TFs interact with multiple cis-regulatory elements across the CFTR locus implicating a more global role in intestinal expression of the gene.