Objective: To investigate the effects of epidermal growth factor receptor (EGFR) inhibitor AG1478 in combination with 5-fluorouracil (5-FU) on proliferation, migration and invasiveness of cholangiocarcinoma cells in vitro. Methods: Human cholangiocarcinoma QBC939 cells were cultured in vitro and exposed to AG1478 (10 μmol/L) and 5-FU (100 mg/L) alone or in combination for 24 h. The cell proliferation, migration and invasiveness were measured by CCK-8 assay, wound-scratch assay and Matrigel invasion chamber assay, respectively. Results: The abilities of proliferation, migration and invasion of QBC939 cells were significantly decreased after either lone AG1478 or 5-FU treatment (all P<0.05), and above effects were further enhanced by the combined treatment compared with either lone drug treatment (all P<0.05). Conclusion: Combination use of EGFR inhibitor and 5-FU may be an effective method for inhibiting the growth, migration and invasiveness of cholangiocarcinoma cells.