The underlying defect in cystic fibrosis is mutation of the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel expressed at the apical surface of lung epithelia. In addition to its export and maintenance at the cell surface, CFTR regulation involves repeated cycles of transport through the endosomal trafficking system, including endocytosis and recycling. Many of the known disease mutations cause CFTR intracellular trafficking defects that result in failure of ion channel delivery to the apical plasma membrane. Corrective maneuvers directed at improving transport to the plasma membrane are thwarted by rapid internalization and degradation of the mutant CFTR proteins. The molecular mechanisms involved in these processes are not completely understood but may involve protein-protein interactions with the C-terminal type I PDZ-binding motif of CFTR. Using a proteomic approach, we identify sorting nexin 27 (SNX27) as a novel CFTR binding partner in human airway epithelial Calu-3 cells. SNX27 and CFTR interact directly, with the SNX27 PDZ domain being both necessary and sufficient for this interaction. SNX27 co-localizes with internalized CFTR at sub-apical endosomal sites in polarized Calu-3 cells, and either knockdown of the endogenous SNX27, or over-expression of a dominant-negative SNX27 mutant, resulted in significant decreases in cell surface CFTR levels. CFTR internalization was not affected by SNX27 knockdown, but defects were observed in the recycling arm of CFTR trafficking through the endosomal system. Furthermore, knockdown of SNX27 in Calu-3 cells resulted in significant decreases in CFTR protein levels, consistent with degradation of the internalized pool. These data identify SNX27 as a physiologically significant regulator of CFTR trafficking and homeostasis in epithelial cells.