Abstract: OBJECTIVE To explore the effect of dihydroartemisinin @ carbon dot nanoparticles(CDs@DHA) on killing Acanthamoeba castellanii and observe the action mechanisms. METHODS CDs@DHA nanoparticles were prepared via supramolecular co-assembly by using carbon dots (CDs) as carriers. The uptake capacities of trophozoites and cysts were assessed through drug uptake assays. The cells were subjected to trypan blue staining to calculate the killing rate after exposure to various concentrations of DHA and CDs@DHA, and the morphologic changes were examined by electron microscopy. RESULTS The uptake of CDs@DHA was increased with the time (1 hour, 2 hours, and 4 hours), the intake of the trophozoites was higher than that of the cysts. The mortality rate of trophozoites was remarkably higher in the DHA and CDs@DHA group than in the control group after the exposure to 12.5, 25 and 50 μmol/L for 4 hours(P＜0.05), and the lethal effect of the CDs@DHA was remarkably higher than that of the DHA at the concentrations of 25 and 50 μmol/L (P＜0.05). CDs@DHA exhibited remarkable concentration -dependent property to the lethal effect on trophozoites (P＜0.05). The mortality rate of trophozoites was remarkably higher in the DHA and CDs@DHA groups than in the control group after the exposure to low, medium, and higher concentrations of drugs for 6 and 12 hours(P＜0.05), and the lethal effect of the CDs@DHA was remarkably higher than that of the DHA at the same concentration (P＜0.05), the CDs@DHA showed remarkable concentration-dependent property to the lethal effect on trophozoites (P＜0.05). The mortality rate of cysts was remarkably higher in the DHA and CDs@DHA groups than in the control group after their exposure to them at the concentrations of 50, 100 and 200 μmol/L for 24 and 48 hours(P＜0.05), and the mortality rate of the CDs@DHA group was higher than that of the DHA group at the same concentration (P＜0.05). CDs@DHA showed remarkable concentration-dependent property to the lethal effect on the cysts (P＜0.05). The result of the electron microscopy revealed that CDs@DHA induced shrinkage and disruption of the trophozoite cell membrane along with severe damage to organelles and resulted in disappearance of the wall structure and marked internal pyknosis of the cysts; overall, it caused more remarkable damage than DHA. CONCLUSIONS CDs@DHA can remarkably enhance the killing efficacy against both Acanthamoeba trophozoites and cysts by efficiently penetrating the cyst wall and disrupting amoebic structures, laying a solid foundation for the development of novel therapeutics and diagnostic agents against Acanthamoeba infections.