Background and Aims The mitotic kinase Aurora-B is considered to be a proto-oncogene of cholangiocarcinoma. The action of centrosome protein U (CENPU) in mitosis is governed by phosphorylation regulation of Aurora-B kinase. The previous study conducted by the authors’ group has identified CENPU in cholangiocarcinoma and confirmed that the expression level of CENPU is significantly higher in cholangiocarcinoma tissue than that in its corresponding adjacent tissue. So, it is speculated that Aurora-B probably participates the tumor biological process of cholangiocarcinoma through regulation of the CENPU phosphorylation. Therefore, this study was performed to investigate the relationship between Aurora-B and CENPU and their effects in cholangiocarcinoma.Methods The expressions of Aurora-B and CENPU as well as the phosphorylated CENPU (p-CENPU) in 10 pairs of specimens of cholangiocarcinoma and para-carcinoma tissue were determined by immunohistochemical staining. The expressions of Aurora-B and CENPU and their correlation in cholangiocarcinoma were analyzed in TCGA database. The cholangiocarcinoma QBC939 cells with Aurora-B gene knockout were constructed, and then, the expressions of CENPU and p-CENPU were determined by Western blot analysis and the proliferative activity was detected by CCK8 assay. QBC939 cells with mutated phosphorylation site of CENPU were constructed, in which, the changes in expressions of Aurora-B, CENPU and p-CENPU as well as the proliferation activity were observed.Results The results of immunohistochemical staining showed that positive expression rates of Aurora-B and CENPU in cholangiocarcinoma tissue were 22.61% and 12.34%, respectively, but nearly none of them expressed in adjacent tissue. Meanwhile, the expression level of p-CENPU in cholangiocarcinoma tissue was remarkably higher than that in adjacent tissue. TCGA data showed that both expressions of Aurora-B and CENPU were up-regulated in cholangiocarcinoma (both P<0.05), and there was a positive correlation between the expressions of Aurora-B and CENPU (r=0.7322, P<0.05). In QBC939 cells after knockdown of Aurora-B expression, there was no significant change in CENPU expression, but the p-CENPU expression was down-regulated, and the proliferation activity was significantly decreased (P<0.05). In QBC939 cells after mutating the phosphorylation site of CENPU or adding Aurora-B inhibitor, Aurora-B expression showed no change in the former, but was significantly reduced in the latter, and in both conditions, the CENPU expressions showed no changes, but the p-CENPU expressions were decreased; after mutating the phosphorylation site of CENPU, the proliferation activity of QBC939 cells mediated by Aurora-B was significantly lower than that of the wild-type (P<0.05).Conclusion CENPU is the phosphorylation substrate of Aurora-B in cholangiocarcinoma cells. Aurora-B promotes the proliferation of cholangiocarcinoma cells probably by phosphorylation of CENPU.