Increased intestinal permeability plays a crucial role in the pathophysiology of inflammatory bowel disease (IBD). Select IBD candidate genes are associated with increased intestinal permeability and/or altered mucosal immune responses. Loss-of-function mutations in the protein tyrosine phosphatase non-receptor type 2 (PTPN2) gene are associated with IBD but their functional contribution to IBD pathogenesis is unknown. We identified a novel role for PTPN2 in protecting epithelial barrier function in vitro and in vivo. The aim of this study was to identify if barrier defects in Ptpn2-deficient mice are associated with altered production of barrier-modifying inflammatory cytokines in different intestinal regions. Methods: In vivo intestinal permeability was assessed by oral gavage of 4 kD FITC-dextran (FD4) to 18-21 day old Ptpn2 wild-type (WT), heterozygous (Het) and knockout (KO) mice (male and female co-housed littermates) and serum collection after 4 hrs to quantify FD4. Ileum, cecum, proximal and distal colon were isolated and mounted in Ussing chambers for ex vivo studies of barrier function (FD4 permeability and transepithelial electrical resistance (TER)). Tight junction protein analysis was performed by immunohistochemistry and immunofluorescence confocal microscopy. Serum and intestinal cytokine levels were determined by Multiplex ELISA analysis. Results: In vivo FD4 permeability was significantly increased in Het vs. WT (2.0 ± 0.1 fold above WT; p<0.001 by ANOVA; n=5) and further elevated in KO vs. WT (3.5 ± 0.5 fold; p<0.001; n=5). Ex vivo intestinal tissues showed increased FD4 flux and reduced TER in multiple regions in Ptpn2 KO mice compared with WT mice, while Het mice exhibited barrier defects but to a lesser extent than KO mice (n=5-11). This was consistent with increased intestinal claudin-2 expression in Het and KO mice and mislocalization of ZO-1 and occludin vs. WT (n=2). IFN-gamma, TNF-alpha and IL-6 levels were increased in KO mouse serum and large intestine with the increase in IFN-gamma in KO mouse serum (460 ± 260 vs. 3 ± 0.1 pg/ml in WT; mean ± SEM; n=3 in triplicate) and cecum (142 ± 42 vs. 9 ± 1 pg/ml in WT; mean ± SEM; n=2 performed in triplicate) most prominent. These cytokines were increased in Het mice in select tissue regions vs. WT (n=2-3). Conclusion: Reduced expression of PTPN2 leads to compromised intestinal barrier function and elevated expression of inflammatory cytokines known to promote epithelial barrier defects. These data indicate additional mechanisms by which PTPN2 loss of function may contribute to decreased intestinal barrier properties relevant to IBD pathogenesis.