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Cytarabine (Ara-C) is a widely used drug in acute myeloid leukemia (AML). However, it faces serious challenges in clinical application due to serious side effects such as gastrointestinal disorders and neurologic toxicities. Until now, the mechanism of Ara-C-induced damage is not clear. Here, we used Drosophila melanogaster (fruit fly) as the in vivo model to explore the side effects and mechanism of Ara-C. Our results showed that Ara-C supplementation delayed larval development, reduced lifespan, impaired locomotor capacity, and increased susceptibility to stress response in adult flies. In addition, Ara-C led to the intestinal morphological damage and ROS accumulation in the guts. Moreover, administration of Ara-C promoted gene expressions of Toll pathway, IMD pathway, and apoptotic pathway in the guts. These findings raise the prospects of using Drosophila as in vivo model to rapidly assess chemotherapy-mediated toxicity and efficiently screen the protective drugs.