The alteration of the intestinal barrier function is currently believed to be involved in the pathogenesis of gut diseases mainly associated with the activation of inflammation processes.Diet plays an important role in the control of human gut integrity. Theobromine is a natural methylxanthine present in dark chocolate particularly abundant in cocoa bean shell. This is a polyphenol rich by-product generated in cocoa industrial processing, which is gaining value as a functional ingredient. This study aims to highlight for the first time the capability of theobromine in protecting the intestinal cell monolayer from a mixture of dietary oxysterols showing an inflammatory action in terms of IL-8 and MCP-1 overproduction. Differentiated CaCo-2 cells were treated with 60 μM oxysterol mixture and pre-incubated with 10 μM theobromine. Intestinal barrier damage was investigated in terms of tight junction claudin 1, occludin and JAM-A protein levels, matrix metalloproteinase (MMP) −2 and −9 activation and anti/pro-apoptotic protein changes.The observed cell monolayer permeability protection by theobromine may be due to its ability to inhibit the production of cytokines and MMPs that can be responsible for tight junction loss and apoptosis in intestinal cells. Our findings provide additional mechanistic hints on the healthy effect of theobromine cocoa component as an attractive natural molecule in the prevention of inflammatory gut diseases.