The global impact of nanoplastics (NPs) as emerging pollutants on water environments remains uncertain. Previous studies have focused on the toxicity of NPs itself, often ignoring the important effects of exposure routes. Here, we evaluated the impact of different exposure routes (waterborne, foodborne, and coexposure) of 80 nm polystyrene NPs on intestinal accumulation and inflammatory response using zebrafish. Our results demonstrated that aqueous and dietary exposures led to both time-and dose-dependent bioaccumulation of NPs, damaged intestinal structure, and induced immune cells and genes in the intestine. While waterborne exposure caused lower NPs accumulation than foodborne exposure, both demonstrated similar immunotoxicity in the intestine. In addition, co-exposure can lead to a higher accumulation of NPs and toxicity in zebrafish, suggesting that the risk of environmental exposure may be underestimated, as current toxicity assessments have focused on single-route exposure scenarios. We further revealed that NPs activated downstream genes of the toll-like 4 receptor, suggesting that NPs can cause an inflammatory response. Our study is the first known to report that exposure to NPs via different routes induces an accumulation in the intestine and can cause similar intestinal disturbance effects, providing new insights for adequately assessing the potential risks of NPs.