Considering that the prognosis of patients with advanced biliary tract cancer (BTC) remains very poor, with a median survival of less than 1 year, new therapeutic approaches need to be developed. In the present study, a phase II clinical trial of personalized peptide vaccination (PPV) was conducted in advanced BTC patients to evaluate the feasibility of this treatment and to identify potential biomarkers. A maximum of 4 human leukocyte antigen-matched peptides, which were selected based on the pre-existing host immunity prior to vaccination, were subcutaneously administered (weekly for 6 consecutive weeks and bi-weekly thereafter) to 25 advanced BTC patients without severe adverse events. Humoral and/or T cell responses specific to the vaccine antigens were substantially induced in a subset of the vaccinated patients. As shown by multivariate Cox regression analysis, lower interleukin-6 (IL-6) and higher albumin levels prior to vaccination and greater numbers of selected vaccine peptides were significantly favorable factors for overall survival [hazard ratio (HR)=1.123, 95% confidence interval (CI) 1.008-1.252, P=0.035; HR=0.158, 95% CI 0.029-0.860, P=0.033; HR=0.258, 95% CI 0.098-0.682, P=0.006; respectively]. Based on the safety profile and substantial immune responses to vaccine antigens, PPV could be a promising approach for refractory BTC, although its clinical efficacy remains to be investigated in larger-scale prospective studies. The identified biomarkers are potentially useful for selecting BTC patients who would benefit from PPV.