Objective: To investigate the expressions of mucin-3 (MUC3) and -4 (MUC4) in tissues of intrahepatic cholangiocarcinoma (ICC) associated with hepatolithiasis and their clinical significance. Methods: The MUC3 and MUC4 expressions in the 24 specimens of normal bile duct tissue, 44 specimens of bile duct tissue from hepatolithiasis patients, and 38 specimens of hepatolithiasis-associated ICC were determined by immunohistochemical staining. The relations of MUC3 and MUC4 expressions with clinical factors and prognosis of the patients with hepatolithiasis-associated ICC were analyzed. Results: The positive expression rates of both MUC3 and MUC4 were significantly different in the three different tissues (both P<0.05); in normal bile duct tissue, bile duct tissue from hepatolithiasis patients and hepatolithiasis-associated ICC tissue, the positive MUC3 expression rates were ranked in a decreasing order (79.2%, 56.8% and 36.8%), while the opposite was true for MUC4 (29.2%, 79.5% and 86.8%). In patients with hepatolithiasis-associated ICC, both MUC3 and MUC4 expressions were significantly related to the tumor histological differentiation and the presence or absence of lymph node metastasis, and MUC4 expression was also significantly related to the presence or absence of portal vein invasion (all P<0.05). In patients with hepatolithiasis-associated ICC, the postoperative survival rate in patients with positive MUC3 expression was significantly higher than that in patients with negative MUC3 expression, and in patients with positive MUC4 expression was significantly lower than that in patients with negative MUC4 expression (all P<0.05). Conclusion: The MUC3 expression is decreased and the MUC4 expression is increased in hepatolithiasis-associated ICC tissue, and the changes of their expressions are closely related to the development, invasion and metastasis of hepatolithiasis-associated ICC.