Numb, an endocytic protein, is involved in both neural differentiation and protein post-endocytic trafficking. Although negative Numb expression has been linked to human mammary carcinomas, little is known about its expression and functions in other diseases. In the present study, we observed that Numb is expressed in renal tubule epithelia and its expression is increased in the fibrotic kidney in vivo. We determined that in proximal tubular epithelial cells (NRK52E cells), TGF-β1 induces the expression of Numb and ectopic expression of Numb leads to dissolution of E-cadherin adhesion, reorganization of cytoskeleton, activation of Rac1 and Cdc42, and enhancement of migration. Either knockdown of α-adaptin or overexpression of Numb asparagine-proline-phenylalanine (NPF) mutant interferes with AP-2 dependent endocytosis and rescues Ecadherin level in NRK52E cells. Moreover, knockdown of integrin β1 or α-adaptin, and overexpression of a Numb dominant-negative form (Numb phosphotyrosine binding [PTB] domain) impair integrin endocytosis, and markedly inhibit Numb-induced cell migration and activation of Rac1 and Cdc42. Taken together, our work identifies Numb as an important player in renal fibrosis, by regulating epithelial-to-mesenchymal transition (EMT) process including E-cadherin adhesion dissolution, actin reorganization, and migration enhancement in NRK52E cells.