Recent studies have linked impairment of intestinal epithelial function in inflammatory bowel disease to the disturbance of endoplasmic reticulum homeostasis (ER) in response to stress. Most studies are on goblet and Paneth cells, which are considered more susceptible to stress due to their role in the protection of intestinal epithelium against microbes and harmful substances. However, studies on the role of inflammation-induced ER stress in absorptive intestinal cells are scarce. In this study, we show, using Caco-2 cells as a model of intestinal epithelial barrier, that inducing ER stress using a cocktail mixture of pro-inflammatory mediators [TNFα (50 ng/ml), MCP1 (50 ng/ml), and IL-1β (25 ng/ml)] as observed in IBD patients induces ER stress and leads to significant changes in key proteins of the apical (sucrase-isomaltase (SI), dipeptidyl-peptidase (DPPIV), and ezrin) and basolateral (E-cadherin, zonula occludens (ZO-1), and connexin-43) membranes. Aberrant trafficking of SI, DPPIV was observed as early as 8 h post-inflammation-induced ER stress and even in the absence of loss of intestinal cell integrity. The observed effect was associated with a re-localization of ezrin, ZO-1, and connexin-43, key differentiation and junction proteins. Collectively, this study shows that disruption of the trafficking of key digestive enzymes of the intestinal epithelium occur in response to inflammation induced ER stress before the loss of monolayer integrity.