The effectiveness and stability of epithelial barrier depend on apical junctional complexes (AJCs), which consist of tight junctions (TJs) and adherens junctions (AJs). E-cadherin is the primary component of AJs and it is essential for maintenance of cell-to-cell interactions and regulates the epithelial barrier. However, the exact mechanism underlying E-cadherin expression, particularly at the post-transcriptional level, remains largely unknown. RNA-binding proteins CUG-binding protein 1 (CUGBP1) and HuR are highly expressed in the intestinal epithelial tissues and modulate the stability and translation of target mRNAs. Here, we present evidence that CUGBP1 and HuR interact directly with the 3'-untranslated region (3'-UTR) of E-cadherin mRNA and regulate E-cadherin translation. CUGBP1 overexpression in Caco-2 cells inhibited E-cadherin translation by increasing the recruitment of E-cadherin mRNA to processing bodies (PBs), thus resulting in an increase in paracellular permeability. Overexpression of HuR exhibited an opposite effect on E-cadherin expression by preventing the translocation of E-cadherin mRNA to PBs, therefore prevented CUGBP1-induced repression of E-cadherin expression. Elevation of HuR also abolished the CUGBP1-induced epithelial barrier dysfunction. These findings indicate that CUGBP1 and HuR negate each other's effects in regulating E-cadherin translation by altering the recruitment of E-cadherin mRNA to PBs and play an important role in the regulation of intestinal barrier integrity under various pathophysiological conditions.