The Crohn's disease candidate gene, protein tyrosine phosphatase nonreceptor type 2 (PTPN2), has been shown to regulate epidermal growth factor (EGF)-induced phosphatidylinositol 3-kinase (PI3K) activation in fibroblasts. In intestinal epithelial cells (IECs), EGF-induced EGF receptor (EGFR) activation and recruitment of PI3K play a key role in regulating many cellular functions including Ca(2+)-dependent Cl(-) secretion. Moreover, EGFR also serves as a conduit for signaling by other non-growth factor receptor ligands such as the proinflammatory cytokine, IFN-γ. Here we investigated a possible role for PTPN2 in the regulation of EGFR signaling and Ca(2+)-dependent Cl(-) secretion in IECs. PTPN2 knockdown enhanced EGF-induced EGFR tyrosine phosphorylation in T(84) cells. In particular, PTPN2 knockdown promoted EGF-induced phosphorylation of EGFR residues Tyr-992 and Tyr-1068 and led subsequently to increased association of the catalytic PI3K subunit, p110, with EGFR and elevated phosphorylation of the downstream marker, Akt. As a functional consequence, loss of PTPN2 potentiated EGF-induced inhibition of carbachol-stimulated Ca(2+)-dependent Cl(-) secretion. In contrast, PTPN2 knockdown affected neither IFN-γ-induced EGFR transactivation nor EGF- or IFN-γ-induced phosphorylation of ERK1/2. In summary, our data establish a role for PTPN2 in the regulation of EGFR signaling in IECs in response to EGF but not IFN-γ. Knockdown of PTPN2 directs EGFR signaling toward increased PI3K activation and increased suppression of epithelial chloride secretory responses. Moreover, our findings suggest that PTPN2 dysfunction in IECs leads to altered control of intestinal epithelial functions regulated by EGFR.