The purpose of our study was to investigate anticancer activity of arsenic trioxide (As2O3) on cholangiocarcinoma through in vitro and in vivo experiments using human cholangiocarcinoma cancer cells (CC-t6 cells) and a nude mouse model. The effect of As2O3 on CC-t6 cell survival was determined in vitro using MTT assay. Analysis of cell cycle phase distribution and quantification of apoptosis/necrosis, which were achieved by flow cytometry, were performed in order to understand the mechanism of As2O3. In vivo experiment was performed to assess the effectiveness of local injection of As2O3 on tumor inhibition by comparing the following three groups each consisting of five nude mouse xenograft models: high dose As2O3 (5 mg/kg), low dose As2O3 (1 mg/kg), and saline. In MTT assay, As2O3 inhibited the growth of CC-t6 cells more effectively than cisplatin or adriamycin at concentrations between 1 and 100 μM for most time points between 24 and 72 h (p < 0.05). With increased concentration of As2O3, there was dose-dependent increase in G 0/G 1 phase and dose-dependent decrease in S phase. As2O3-mediated inhibition of cell viability was achieved via induction of apoptosis and necrosis in a dose-dependent manner. Injection of As2O3 into CC-t6-induced tumors in nude mice inhibited the growth of subcutaneous tumor xenografts. As2O3 treatment dose-dependently inhibited the proliferation of CC-t6 cells via G 0/G 1 phase arrest and retarded tumor growth in nude mice, suggesting that As2O3 may be effective in the treatment of cholangiocarcinoma.