Objective: To investigate the expressions of annexin A2 and the epithelial-mesenchymal transition (EMT) markers E-cadherin and vimentin in hepatolithiasis-associated intrahepatic cholangiocarcinoma and their significance. Methods: The expressions of annexin A2, E-cadherin and vimentin in tumor tissues from 46 patients with hepatolithiasis-associated intrahepatic cholangiocarcinoma (tumor group), bile duct tissues with chronic inflammation from 50 patients with simple hepatolithiasis (inflammation group) and normal bile duct tissues form 35 patients undergoing surgical resection for hepatic hemangioma or liver injury (normal group) were determined by immunohistochemical staining. The expressions of the three proteins among the groups were compared and their relations with clinicopathologic factors and prognosis of the patients with hepatolithiasis-associated intrahepatic cholangiocarcinoma were analyzed. Results: The positive expression rates of both annexin A2 and vimentin were presented in descending order as tumor group>inflammation group>normal group (69.6% vs. 36.0% vs. 11.4%; 54.3% vs. 28.0% vs. 8.6%, all P<0.05), while the opposite pattern was seen in that of E-cadherin (21.7% vs. 48.0% vs. 71.4%, all P<0.05). In cholangiocarcinoma tissues, there was a positive correlation between the annexin A2 and vimentin expression (r=0.627, P<0.05), and negative correlation between the E-cadherin expression and either the annexin A2 or vimentin expression (r=–0.682; r=–0.575, both P<0.05). The expressions of annexin A2 and vimentin were significantly associated with the degree of tumor differentiation, lymph node metastasis and TNM stage, and E-cadherin expression was significantly associated with the degree of tumor differentiation and lymph node metastasis (all P<0.05). Among the patients with hepatolithiasis-associated intrahepatic cholangiocarcinoma, the survival rate in cases with positive annexin A2 or vimentin expression was significantly lower than that in their corresponding negative ones, and the survival rate in cases with negative E-cadherin expression was significantly lower than that in those with its positive expression (all P<0.05). Conclusion: The annexin A2/EMT pathway may probably play an important role in the development and progression of hepatolithiasis-associated intrahepatic cholangiocarcinoma. The expressions of annexin A2, E-cadherin and vimentin are closely related to the degree of malignancy of the tumor and prognosis of the patients.