The aim of this study was to determine the level of expression of Mina53 in human cholangiocarcinoma (CCA) and to explore the role of Mina53 in carcinogenesis and tumor progression and its clinical significance in CCA. The level of expression of Mina53, p53 and Ki67 was investigated by immunohistochemistry in 69 surgically resected CCA tissues and 21 adjacent non-cancerous tissues. The correlation between Mina53 expression and clinicopathological characteristics and the expression of p53 and Ki67 was examined. Positive expression of Mina53 was observed in 61 of 69 CCA cases (88.4%) and 1 case (4.8%) of adjacent non-cancerous tissue. The level of expression of Mina53 in CCA was markedly higher than in the adjacent non-cancerous tissues. An increased level of expression of Mina53 in CCA was significantly associated with histological differentiation (P<0.01), TNM stage (P<0.05) and lymph node metastasis (P<0.01). There was no significant correlation between the level of Mina53 expression and gender, age or distant metastasis (P>0.05). However, the expression of Mina53 was associated with the expression of p53 in CCA (P<0.05). In addition, increased levels of expression of Mina53 in CCA were positively associated with Ki67 levels (r=0.801, P<0.01, as calculated by association analysis). Therefore, the upregulation of Mina53 expression may be significant in the carcinogenesis and development of human CCA and could have significant clinical value.