Fluorouracil (5-FU) is thefirst-line chemotherapeutic drug for cholangiocarcinoma (CCA), but its efficacy has beencompromised by the development of resistance. Development of 5-FU resistance is associated with elevatedexpression of its cellular target, thymidylate synthase (TYMS). E2F1 transcription factor has previously been shown tomodulate the expression of FOXM1 and TYMS. Immunohistochemical (IHC) analysis revealed a strong correlatedupregulation of FOXM1 (78%) and TYMS (48%) expression at the protein levels in CCA tissues. In agreement, RT-qPCRand western blot analyses of four human CCA cell lines at the baseline level and in response to high doses of 5-FUrevealed good correlations between FOXM1 and TYMS expression in the CCA cell lines tested, except for the highly 5-FU-resistant HuCCA cells. Consistently, siRNA-mediated knockdown of FOXM1 reduced the clonogenicity and TYMSexpression in the relatively sensitive KKU-D131 but not in the highly resistant HuCCA cells. Interestingly, silencing ofTYMS sensitized both KKU-D131 and HuCCA to 5-FU treatment, suggesting that resistance to very high levels of 5-FU isdue to the inability of the genotoxic sensor FOXM1 to modulate TYMS expression. Consistently, ChIP analysis revealedthat FOXM1 binds efficiently to the TYMS promoter and modulates TYMS expression at the promoter level upon 5-FUtreatment in KKU-D131 but not in HuCCA cells. In addition, E2F1 expression did not correlate with either FOXM1 orTYMS expression and E2F1 depletion has no effects on the clonogenicity and TYMS expression in the CCA cells. Inconclusion, our data show that FOXM1 regulates TYMS expression to modulate 5-FU resistance in CCA and that severe5-FU resistance can be caused by the uncoupling of the regulation of TYMS by FOXM1. Ourfindings suggest that theFOXM1–TYMS axis can be a novel diagnostic, predictive and prognostic marker as well as a therapeutic target for CCA