Mutations in CFTR lead to dysfunction of tubular organs, which is currently attributed to impairment of its conductive property. We now show that CFTR regulates tight junction (TJ) assembly and epithelial cell differentiation via modulation of the ZO-1/ZONAB pathway. CFTR co-localizes with ZO-1 in TJs of trachea and epididymis, and is expressed before ZO-1 in Wolffian ducts. CFTR interacts with ZO-1 via its PDZ binding domain. In a 3D epithelial cell culture model, CFTR regulates TJ assembly and is required for tubulogenesis. CFTR inhibition or knockdown reduces ZO-1 expression and induces the translocation of the transcription factor ZONAB from TJs to the nucleus, followed by up-regulation of CCND1 and down-regulation of ErbB2. The epididymal tubules of cftr−/− and cftrΔF508 mice have reduced ZO-1 levels, increased ZONAB nuclear expression, and decreased epithelial cell differentiation, illustrated by the reduced expression of apical AQP9 and V-ATPase. This study provides a new paradigm for the etiology of diseases associated with CFTR mutations, including cystic fibrosis.