Objective: To investigate the expressions and clinical significance of high mobility group protein box l (HMGBl) and NF-κB in tissues of hepatolithiasis-associated intrahepatic cholangiocarcinoma. Methods: The expressions of HMGB1 and NF-κB in tumor tissues from 40 cases of hepatolithiasis-associated intrahepatic cholangiocarcinoma (tumor group), inflammatory bile duct tissues from 40 cases of simple hepatolithiasis (inflammatory group) and normal bile duct tissues form 30 cases undergoing surgical resection for hepatic hemangioma or liver injury (normal group) were determined by immunohistochemical staining. The difference in expressions of the two proteins among the tissues was compared and their relations with clinicopathologic factors and prognosis of the patients with hepatolithiasis-associated intrahepatic cholangiocarcinoma were analyzed. Results: Both expression intensities of HMGB1 and NF-κB among the different tissues were statistically different, which were increased in the order of normal group, inflammatory group, and tumor group,respectively (all P<0.05); there was a positive correlation between the expressions of HMGB1 and NF-κB in cholangiocarcinoma tissue (χ2=13.713, r=0.586, P<0.05). The HMGB1 expression was significantly associated with the degree of tumor differentiation, the depth of tumor invasion and the lymph node metastasis in patients with hepatolithiasis-associated intrahepatic cholangiocarcinoma (all P<0.05), while the NF-κB expression showed no significant relation with any of the selected factors (all P>0.05). In patients with hepatolithiasis-associated intrahepatic cholangiocarcinoma, the survival rate in cases with positive HMGB1 expression was significantly lower than those with negative HMGB1 expression (P<0.05), however, the NF-κB expression exerted no impact on survival (P>0.05). Conclusion: The HMGB1/NF-κB pathway probably participate in the process of occurrence and development of hepatolithiasis-associated intrahepatic cholangiocarcinoma, and HMGB1 expression may exert a decisive impact on degree of malignancy of the tumor and prognosis of the patients.