Vasoactive Intestinal Peptide (VIP) is a topical airway gland secretagogue regulating fluid secretions, primarily by stimulating CFTR-dependent chloride secretion that contributes to the airways innate defense mechanism. We had previously reported that prolonged VIP stimulation of VPAC1 receptor in airway cells enhances CFTR function by increasing its membrane stability. In the present study, we have identified the key effectors in the VIP signaling cascade in the human bronchial serous cell line Calu-3. Using immunocytochemistry and in-situ proximity ligation assays, we found that VIP stimulation increased CFTR membrane localization by promoting its co-localization and interaction with the scaffolding protein NHERF1, a PDZ protein known as a positive regulator for CFTR membrane localization. VIP stimulation also increased phosphorylation, by PKC, of the actin-binding protein complex ERM (Ezrin-Radiaxin-Moesin) and its interaction with NHERF1 and CFTR complex. On the other hand, it reduced intracellular CFTR co-localization and interaction with CAL (CFTR Associated ligand), another PDZ protein known to compete with NHERF1 for CFTR interaction, inducing cytoplasmic retention and lysosomal degradation. Reducing NHERF1 or ERM expression levels by specific siRNAs prevented the VIP effect on CFTR membrane stability. Furthermore, iodide efflux assays confirmed that NHERF1 and P-ERM are necessary for VIP regulation of the stability and sustained activity of membrane CFTR. This study shows the cellular mechanism by which prolonged VIP stimulation of airway epithelial cells regulates CFTR-dependent secretions.