The mechanisms underlying the association between intestinal barrier dysfunction and type 2 diabetes (T2DM) remain unclear. Endoplasmic reticulum (ER) stress activation plays a pivotal role in glucotoxicity-mediated cellular damage, and it has been found to affect gut barrier integrity. Herein, we investigated whether subjects with prediabetes and T2DM display activation of ER stress in the gut along with an altered barrier integrity, and whether hyperglycemia directly drives these aberrations. We also tested the positive effects of the ER stress inhibition by the chemical chaperone 4-phenylbutyrate (4-PBA) on diabetes-related intestinal barrier dysfunction. To this end, we evaluated levels of the ER stress markers and the tight junction (TJ) proteins in colonic mucosa biopsies of 55 individuals stratified according to their glucose tolerance in NGT, prediabetes and T2DM. Colonic mucosa samples collected from NGT subjects were cultured in presence/absence of high glucose (25mM-50mM), and those from subjects with altered glucose tolerance were cultured in presence/absence of 4-PBA 10mM. As compared to NGT group, individuals with prediabetes and T2DM exhibited higher colonic levels of the ER stress markers IRE-1α, phospho-eIF2α and of the pro-apoptotic/pro-inflammatory factors CHOP, cleaved PARP and phospho-JNK, coupled with lower TJ ZO-1 and occludin abundance. HG exposure increased IRE-1α, phospho-eIF2α, CHOP, cleaved PARP and phospho-JNK levels, and significantly reduced ZO-1 and occludin abundance in the colonic mucosa. Next, colonic mucosa biopsies obtained from prediabetic or T2DM subjects were cultured in absence/presence of the ER stress inhibitor 4-PBA. Treatment with 4-PBA inhibited ER stress-related response in the gut, and concomitantly counteracted the activation of pro-inflammatory/pro-apoptotic pathways and increased TJ protein levels. We demonstrate that hyperglycemia directly promotes ER stress activation and TJ alterations in the colonic mucosa, and ER stress inhibition by 4-PBA treatment counteracts diabetes-related gut barrier damage.