Persistent organic pollutants (POPs) have been widely suggested as contributors to the aquatic insect biomass decline, and their bioavailability is affected by engineered particles. However, the toxicity effects of emerging ionizable POPs mediated by differentially charged engineered nanoparticles on aquatic insects are unknown. In this study, 6:2 chlorinated polyfluoroalkyl ether sulfonate (F-53B, an emerging perfluoroalkyl acid alternative) was selected as a model emerging ionizable POP; the effect of differentially charged nanoplastics (NPs, 50 nm, 0.5 g/kg) on F-53B bioaccumulation and gut toxicity to Chironomus kiinensis were investigated through histopathology, biochemical index, and gut microbiota analysis. The results showed that when the dissolved concentration of F-53B remained constant, the presence of NPs enhanced the adverse effects on larval growth, emergence, gut oxidative stress and inflammation induced by F-53B, and the enhancement caused by positively charged NP-associated F-53B was stronger than that caused by the negatively charged one. This was mainly because positively charged NPs, due to their greater adsorption capacity and higher bioavailable fraction of associated F-53B, increased the bioaccumulation of F-53B in larvae more significantly than negatively charged NPs. In addition, positively charged NPs interact more easily with gut biomembranes and microbes with a negative charge, further increasing the probability of F-53B interacting with gut biomembranes and microbiota and thereby aggravating gut damage and key microbial dysbacteriosis related to gut health. These findings demonstrate that the surface charge of NPs can regulate the bioaccumulation and toxicity of ionizable POPs to aquatic insects.