The comprehension of the working principles behind the intestinal transepithelial transport is critical in nutrient and drug development research. Within this framework, microfluidic microphysiological platforms are on the verge of overshadowing traditional systems due to their accuracy in replicating key physiological features of the native tissue. Nevertheless, the effects of fluid mechanical stimuli on the selective permeation characteristics of the gut barrier are still unexplored. This is an indispensable feature for designing more biorelevant organ-on-chip models. Here, an intestine-on-chip platform is conceived to mechanically stimulate cells with three different fluid shear stresses and investigate the relative flow-induced changes of molecule transport alongside the resulting epithelial architecture and barrier functionality. Our results reveal that epithelia grown at lower shears exhibit a ∼1.5 higher and faster paracellular permeability while showing a ∼3 times lower and delayed transcellular uptake compared to layers exposed to higher shear stress. This is corroborated by impedance spectroscopy measurements that display altered tight junctional and bilayer resistance, as well as an increased capacitance of the epithelium in response to variations in mechanical stress within the culture. Taken together, these findings advocate that fluid shear stress can serve as mechano-modulator not only for intestinal transport but also for other epithelial cell lines under physiological circumstances.