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Irinotecan has been proven to have anti-tumor effects and is used for chemotherapy in colorectal cancer. It is a prodrug converted by carboxylesterase to form the active metabolite 7-ethyl-10-hydroxy-camptothecin (SN38). To identify the potential molecular function of SN38 in colon cancer, we investigated the gene expression profile of colon cancer cell lines treated with SN38 and tried to reveal critical genes and biological pathways involved in the response of colon cancer cells to SN38 treatment. The analysis indicates that 447 genes (fold change>4, false discovery rate (FDR)<;0.05%) were differentially expressed after SN38 treatment. 464 genes (fold change>2) were predicted to be differentially expressed with exposure time. The expression pattern of 1082 genes (fold change>2) may be cell line-specific. 58 colon related genes were annotated in Gene Ontology and 54 important pathways were found from GeneGO database using enrichment analysis. Pathways involved in cell cycle or apoptosis such as DNA-damage-induced apoptosis, role of Small Ubiquitin-like Modifier (SUMO) in p53 regulation were considered as colon cancer significant pathways. The results demonstrated that some cell cycle and apoptosis pathways were affected by SN38. Our results were generally consistent with previous studies on SN38. Moreover, we employed a more powerful biological pathway database and also obtained other colon cancer related pathways such as signal transduction pathways that were all significant in statistic level. We predicted that these pathways may be important to the SN38 treatment in colon cancer and needed to be further validated in experiment.