Abstract Background and aims Mucin 5AC (MUC5AC) is a glycoprotein found in different epithelial cancers, including biliary tract cancer (BTC). The aims of this study were to investigate role of MUC5AC as serum marker for BTC and its prognostic value after surgery with curative intent. Patients and methods From January 2007 to July 2012 quantitative assessment of serum MUC5AC was performed with ELISA method in a total of 88 subjects. Clinical and biochemical data (including CEA and Ca 19-9) of 49 patients with BTC were compared with a control population including 23 patients with benign biliary disease (BBD) and 16 healthy control subjects (HCS). Results Serum MUC5AC was higher in BTC patients (mean 17.93±10.39 ng/mL) compared to BBD (mean 5.95±5.39 ng/mL) (P < 0.01) and HCS (mean 2.74±1.35 ng/mL) (P < 0.01). Multivariate analysis showed that MUC5AC was significantly related with the presence of BTC compare with Ca 19-9 and CEA P < 0.01, P = 0.080 and P = 0.463, respectively. In BTC group serum MUC5AC ≥ 14 ng/mL was associated with lymph-node metastasis (P = 0.050) and AJCC/UICC stage IVb disease (P = 0.047). Moreover, in patients underwent surgery with curative intent, serum MUC5AC ≥ 14 ng/mL was related to worse prognosis compared with patients with lower levels, with 3-year survival rates of 21.5% and 59.3%, respectively (P = 0.039). Conclusions MUC5AC could be proposed as new serum marker for BTC. Moreover quantitative assessment of serum MUC5AC could be related to tumor stage and long-term survival in patients with BTC underwent surgery with curative intent.