Nanoplastics (NPs, <1 μm) are of great concern worldwide because of their high potential risk toward organisms in aquatic systems, while very little work has been focused on their tissue-specific toxicokinetics due to the limitations of NP quantification for such a purpose. In this study, NPs with two different sizes (86 and 185 nm) were doped with palladium (Pd) to accurately determine the uptake and depuration kinetics in various tissues (intestine, stomach, liver, gill, and muscle) of tilapia (Oreochromis niloticus) in water, and subsequently, the corresponding toxic effects in the intestine were explored. Our results revealed uptake and depuration constants of 2.70-378 L kg day and 0.138-0.407 day for NPs in tilapia for the first time, and the NPs in tissues were found to be highly dependent on the particle size. The intestine exhibited the greatest relative accumulation of both sizes of NPs; the smaller NPs caused more severe damage than the larger NPs to the intestinal mucosal layer, while the larger NPs induced a greater impact on microbiota composition. The findings of this work explicitly indicate the size-dependent toxicokinetics and intestinal toxicity pathways of NPs, providing new insights into the ecological effects of NPs on aquatic organisms.