Histone deacetylation mediated by histone deacetylases (HDACs) has been reported as one of theepigenetic mechanisms associated with tumorigenesis. The poor responsiveness of anticancer drugs found withcholangiocarcinoma (CCA) leads to short survival rate. We aimed to investigate mRNA expression of HDACsclass I and II, and the effect of HDAC inhibitors, suberoylanilide hydroxamic acid (SAHA) and valproic acid(VPA), in CCA in vitro. Expression of HDACs was studied in CCA cell lines (M213, M214 and KKU-100) andan immortal cholangiocyte (MMNK1) by semi-quantitative reverse transcription-PCR. SAHA and VPA, aswell as a classical chemotherapeutic drug 5 -fluorouacil (5-FU) were used in this study. Cell proliferation was determined by sulforhodamine assay. IC50 and IC20 were then analyzed for each agent and cell line. Moreover, synergistic potentional of VPA or SAHA in combination with 5-FU at sub toxic does (IC20) of each agent was alsoevaluated. Statistic difference of HDACs expression or cell proliferation in each experimental condition wasanalyzed by Student’s t-test. The result demonstrated that HDACs were expressed in all studied cell types.Both SAHA and VPA inhibited cell proliferation in a dose-dependent manner. Interestingly, KKU-100 whichwas less senstitive to classical chemotheraoeutic 5-FU was highly was sensitive to HDAC inhibitors. Simultaneous combination of subtoxic doses of HDAC inhibitors and 5-FU signiicantly inhibited cell proliferation in CCA cell lines compared to single sgent treatment(P<0.01), while sequentially combined treatments were less effective.The present study showed inhibitory effects of HDACIs on cell proliferation in CCA cell lines, with synergistic antitumor potential demonstrated by simultaneous combination of VPA or SAHA with 5-FU, suggesting a novel alternative therapeutic strategy in effective treatment of CCA.