The ever-growing production of engineered nanoparticles (NPs) for use in many agricultural, commercial, consumer, and industrial applications will lead to their accidental or intentional release into the environment. Potential routes of environmental exposure include manufacturing or transport spills, disposal of NP‐containing products down the drain and/or in landfills, as well as direct usage on agricultural land. Therefore, NPs will inevitably contaminate aquatic environments and interact with resident organisms. However, there is limited information regarding the mechanisms that regulate NP transport into fish from the environment. Thus, our primary objective was to elucidate the mechanism(s) underlying cellular uptake and intracellular fate of 3–9 nm poly (acrylic acid) NPs loaded with the fluorescent dye Nile red using a rainbow trout (Oncorhynchus mykiss) gill epithelial cell line (RTgill‐W1). In vitro measurements with NP‐treated RTgill-W1 cells were carried out using a combination of laser scanning confocal microscopy, flow cytometry, fluorescent biomarkers (transferrin, cholera toxin B subunit, and dextran), endocytosis inhibitors (chlorpromazine, genistein, and wortmannin), and stains (4′, 6‐diamidino-2-phenylindole, Hoechst 33342, CellMask Deep Red, and LysoTracker Yellow). Clathrin-mediated endocytosis (CME), caveolae‐mediated endocytosis and macropinocytosis pathways were active in RTgill‐W1 cells, and these pathways were exploited by the non-cytotoxic NPs to enter these cells. We have demonstrated that NP uptake by RTgill‐W1 cells was impeded when clathrin-coated pit formation was blocked by chlorpromazine. Furthermore, colocalization analysis revealed a moderate positive relationship between NPs and LysoTracker Yellow-positive lysosomal compartments indicating that CME was the dominant operative mechanism involved in NP internalization by RTgill-W1 cells. Overall, our results clearly show that fish gill epithelial cells internalized NPs via energy‐dependent endocytotic processes. This study enhances our understanding of complex NP‐cell interactions and the results obtained in vitro imply a potential risk to aquatic organisms.