In this study we present the first fish-gut-on-chip model. This model is based on the reconstruction of the intestinal barrier by culturing two intestinal cell lines from rainbow trout, namely epithelial RTgutGC and fibroblastic RTgutF, in an artificial microenvironment. For a realistic mimicry of the interface between the intestinal lumen and the interior of the organism we i) developed ultrathin and highly porous silicon nitride membranes that serve as basement membrane analogues and provide a culture interface for the fish cells; ii) constructed a unique micro-well plate-based microfluidic bioreactor that enables parallelization of experiments and creates realistic fluid flow exposure scenarios for the cells; iii) integrated electrodes in the reactor for non-invasive impedance sensing of cellular well-being. In a first approach, we used this reactor to investigate the response of epithelial fish cells to in vivo-like shear stress rates of 0.002–0.06 dyne per cm2, resulting from fluid flow within the intestinal lumen. Moreover, we investigated the interplay of epithelial and fibroblast cells under optimal flow conditions to carefully evaluate the benefits and drawbacks of the more complex reconstruction of the intestinal architecture. With our fish-gut-on-chip model we open up new strategies for a better understanding of basic fish physiology, for the refinement of fish feed in aquaculture and for predicting chemical uptake and bioaccumulation in fish for environmental risk assessment. The basic principles of our reactor prototype, including the use of ultrathin membranes, an open microfluidic circuit for perfusion and the micro-well plate-based format for simplified handling and avoidance of air-bubbles, will as well be of great value for other barrier-on-chip models.