||Cystic fibrosis transmembrane conductance regulator (CFTR) is a protein associated with the lethal genetic disease cystic fibrosis (CF) and is responsible for electrolyte and water transport in epithelial cells lining various tissues. CFTR functions both as an anion channel, conducting Cl- and HCO3-, and as a regulator of several membrane ion transporters. Recent studies have shown that several proteins bind to the C-terminus of CFTR and affect its cell surface expression and channel gating, but interactions found to date do not account for all the known functions of the CFTR C-terminus, suggesting that as yet unidentified proteins that bind to this region of CFTR modulate the function of the channel. To search for such a protein(s), we performed a yeast two-hybrid screen and identified as a CFTR C-terminus binding protein keratin 18 (K18), an intermediate filament proteins expressed in simple epithelial cells, much like CFTR. Although K18 is traditionally thought to simply provide resilience to mechanical stress in the cell, an emerging body of evidence suggests that K18 may act as a scaffold protein and determine the subcellular localization of other proteins. The interaction of K18 and CFTR was further confirmed by various approaches in mammalian cells. Significantly, our studies on heterologously expressed and endogenous proteins suggested that K18 increased the surface expression of wild-type CFTR. Furthermore, in in vivo study, the absence of CFTR expression and the reduction of CFTR activity were observed in the apical domain of villi of duodenum in K18 knockout mice. In addition, we found that K18 binding site in CFTR is the highly conserved “hydrophobic patch” (F1413LVI), which has been previously reported to be crucial to CFTR stability in plasma membrane with unknown mechanism. Consistent with this, our metabolic pulse chase assays demonstrated that K18 binding increased surface expression of CFTR without any effect on its biosynthesis and maturation. In light of these observations, we concluded that K18 interacts with the hydrophobic patch of CFTR and stabilize CFTR in the apical plasma membrane. These findings offered novel insights into the regulation of CFTR and added a new dimension to our understanding of the function of keratin proteins.