We introduce a novel in vitro rainbow trout intestinal barrier model and demonstrate its suitability for investigating nanoparticle transport across the intestinal epithelium. Rainbow trout (Oncorhynchus mykiss) intestinal cells (RTgutGC) were grown as monolayers on permeable supports leading to a two-compartment intestinal barrier model consisting of a polarized epithelium, dividing the system into an upper (apical) and a lower (basolateral) compartment, and thereby mimicking the intestinal lumen and the portal blood, respectively. The cells express the tight junction protein ZO-1 and build up a transepithelial electrical resistance comparable to the in vivo situation. Fluorescent polystyrene nanoparticles (PS-NPs; average hydrodynamic diameter: 73 ± 18 nm) were accumulated by RTgutGC cells in a time-, temperature- and concentration-dependent manner. Uptake of PS-NPs was confirmed using fluorescence microscopy. Cells formed an efficient barrier largely preventing the translocation of PS-NPs to the basolateral compartment. Taken together, these data demonstrate the suitability of the in vitro barrier model to study the effects of nanoparticles in fish intestinal epithelial cells.