Chemotherapy leads to significant side effects in patients, especially in the gut, resulting in various clinical manifestations and enhanced economic pressure. Until now, many of the underlying mechanisms remain poorly understood. Here, we used Drosophila melanogaster (fruit fly) as in vivo model to delineate the side effects and underlying mechanisms of Irinotecan (CPT-11). The results showed that administration of CPT-11 delayed larval development, induced imbalance of male to female ratio in offspring, shortened lifespan, impaired locomotor ability, changed metabolic capacity, induced ovarian atrophy, and increased excretion. Further, CPT-11 supplementation dramatically caused intestinal damages, including decreased intestinal length, increased crop size, disrupted gastrointestinal acid-based homeostasis, induced epithelial cell death, and damaged the ultrastructure and mitochondria structure of epithelial cells. The cross-comparative analysis between transcriptome and bioinformation results showed that CPT-11 induced intestinal damage mainly via regulating the Toll-like receptor signaling, NF-kappa B signaling, MAPK signaling, FoxO signaling, and PI3K-AKT signaling pathways. In addition, CPT-11 led to the intestinal damage by increasing ROS accumulation. These observations raise the prospects of using Drosophila as a model for the rapid and systemic evaluation of chemotherapy-induced side effects and high-throughput screening of the protective drugs.