Abstract: Trypanosoma sp. is a group of flagellated protozoan parasites that inhabit the blood of various vertebrates, including both freshwater and marine fishes, causing host anemia, organ damage, and even death. As an emerging disease, large yellow croaker trypanosomiasis currently lacks safe and effective therapeutic agents. In this study, using largemouth bass (Micropterus salmoides) as a model host, we systematically evaluated the trypanocidal efficacy and mechanism of action of cepharanthine against Trypanosoma sp. isolated from large yellow croaker through a series of in vitro and in vivo experiments, acute toxicity tests, hematological parameter analysis, histopathological examination, as well as scanning and transmission electron microscopy. Among the eight tested plant-derived compounds, cepharanthine exhibited the strongest in vitro trypanocidal activity, with a median lethal concentration (LC₅₀) of 6.12 mg/L and a 90% lethal concentration (LC₉₀) of 8.75 mg/L against the large yellow croaker-derived Trypanosoma sp., achieving 100% killing rate at 9 mg/L within 4h. In vivo, continuous intragastric administration of cepharanthine at 100 μL (100 mg/kg body weight) daily for 7d significantly reduced blood trypanosome density in largemouth bass and alleviated histopathological damage in the liver, spleen, kidney, and gills. Acute toxicity tests showed that a single intragastric dose of up to 500 mg/kg body weight caused no obvious toxicity to largemouth bass within 96h. Ultrastructural observations revealed that cepharanthine disrupted the integrity of the parasite surface membrane and induced nuclear vacuolation, kinetoplast swelling, and Golgi apparatus dilation. Collectively, cepharanthine demonstrates significant in vitro and in vivo trypanocidal effects against large yellow croaker-derived Trypanosoma sp., and holds potential for development as a novel anti-trypanosomal agent in aquaculture.