The increasing and ubiquitous presence of nanoplastics (NPs) in the aquatic environment demands urgent assessment in order to understand their impact on ecosystems and human health. An important open question is how NPs exposure would interfere with the normal function of the immune system of aquatic organisms. In this context, we aim to understand whether the gut immune homeostasis could be disturbed by NPs exposure. Our previous studies using zebrafish as a model demonstrated that polystyrene nanoplastics (PS-NPs) were efficiently taken up by zebrafish liver (ZFL) cells tending to accumulate in lysosomes, possibly in an attempt of the cell machinery to metabolize PS-NPs. We also observed a modulation of the expression of relevant immune genes in ZFL cells, consecutively exposed to PS-NPs and Poly-(I:C), a viral-like stimulus. In this study, we evaluate the effect of PS-NPs (45 nm) on the immune response of RTgutGC cells (trout gut cells), since fish gastrointestinal epithelium is one of the principal portals of entry for both NPs and pathogens in teleosts. Our results show that: first, PS-NPs do not exert any cytotoxicity on RTgutGC cells; second, PS-NPs are efficiently taken up and accumulate in lysosomes, modulating the number and size of these organelles; third, PS-NPs alone barely affect expression of immune representative genes but trigger a synergic immune response to Poly-(I:C) (viral-like challenge) and LPS (bacterial-like challenge). Hence, these results point out the existence of altered cellular and molecular events that could have a relevant impact on gut mucosal immune response.