The present study was to verify the role of NQO1 on chemotherapeutic response. By suppression of NQO1 using the NQO1 siRNA, it was shown that suppression of NQO1 induced apoptosis and enhanced chemotherapeutic agents (5-fluorouracil (5-FU), gemcitabine (Gem) and doxorubicin (Doxo)) -induced cytotoxicity. In contrast, induction of NQO1 protein expression by NQO1 overexpression vector decreased the drug sensitivity. Nevertheless, the modulatory effects of NQO1 siRNA required the human tumor suppressor gene p53 were associated the increase of p53 and p21 expression. Inhibition of p53 expression by siRNA diminished the chemosensitizing effect of NQO1 siRNA. The result suggested that the chemosensitizing effect was mediated via p53 activation. The association of NQO1 and p53 on chemosensitivity was explored. The present study showed that the treatment of 5-FU and Doxo increased p53 protein while the NQO1 enzyme activity but not the protein was depressed in vitro and in vivo. By the immunoprecipitation assay, NQO1 was found to physically interact with p53, suggesting a protein-protein interaction between p53 and NQO1 leading to suppressed NQO1 activity. The NQO1 inhibition enhanced p53-mediated cell death was decreased only in p53-expressing cancer cells. It was, therefore, suppression of NQO1 activity by drug’s-induced p53 may be partly responsible for chemosensitivity effect. In summary, NQO1 plays a critical role in cytoprotection against chemotherapeutic agents in CCA cells. NQO1 overexpression decreased the drug sensitivity, while inhibition of NQO1 confers the chemosensitizing via p53. Enhanced chemosensitivity by targeting at NQO1 may be a novel treatment strategy to overcome drug resistance in bile duct cancer and other cancers with high NQO1 activity.