In the liver, carboxylesterase (CES) converts irinotecan (CPT-11) to its active metabolite SN-38, which exerts anticancer effects. SN-38 is metabolized to an inactive metabolite SN-38 glucuronide by uridine 5'-diphospho-glucuronosyltransferase 1A1 (UGT1A1). Therefore, single nucleotide polymorphisms (SNPs) of the UGT1A1 gene are responsible for the severe adverse effects associated with the disruption of SN-38 metabolism. However, despite having SNPs of the UGT1A1 gene, many patients metabolize SN-38 sufficiently to avoid severe adverse effects. Among these patients, we found individuals with elevated serum concentrations of hepatocyte growth factor (HGF). The aim of this study was to evaluate whether HGF alters the metabolism of CPT-11, resulting in a reduction in the anticancer effect of CPT11. The cytotoxicity of CPT-11 and SN-38 was evaluated in HepG2 cells pretreated with HGF. Furthermore, we explored the level of expression and mechanisms of activity of CES and UGT1A1. HGF suppressed the cytotoxicity of CPT-11 by decreasing intracellular SN-38 levels that resulted from a decrease in CES2 and an increase in UGT1A1. Furthermore, this HGF-induced suppression was improved by pretreatment with an inhibitor of HGF receptor c-Met, and the improvement was synergistically potentiated by epidermal growth factor receptor (EGFR) inhibitors. Moreover, HGF induced phosphorylation of signal transducer and activator of transcription 3 and transactivated EGFR. These results suggest that HGF is a possible causative agent of acquired clinical resistance in chemotherapy with CPT-11 and could be useful as a predictor of clinical resistance. Additional treatment using c-Met and/or EGFR inhibitors could be a novel strategy to overcome resistance.