Background: For patients with advanced BTC, standard chemotherapy has limited benefit and no molecular targeted agents have been approved. Pembrolizumab is an anti PD-1 immune checkpoint inhibitor which has shown modest activity for advanced BTC patients in prior single-arm phase I/II studies. Considering the heterogeneity of BTC, more data are needed to evaluate the clinical outcomes of pembrolizumab in unresectable or metastatic BTC. Methods: In this prospective cohort study, 39 patients with PD-L1 positive BTC who received pembrolizumab in Asan Medical Center, Seoul, Korea were included (ClinicalTrials.gov identifier, NCT03695952). PD-L1 expression was assessed using immunohistochemistry and PD-L1 positive tumors were defined as the expression of PD-L1 in ≥ 1% of tumor cells. Pembrolizumab was given at a fixed dose of 200 mg intravenously, every 3 weeks. Results: The median age was 61 years old (range, 41-76) and 22 (56.4%) patients were male. Intrahepatic cholangiocarcinoma (CCA) was the most common type (n = 18, 46.2%), followed by gallbladder cancer (n = 12, 30.8%) and extrahepatic CCA (n = 9, 23.1%). Most of the patients had distant metastasis (n = 37, 94.9%). Pembrolizumab was administered as 2nd-, 3rd- and 4th or greater line chemotherapy in 18 (46.2%), 16 (41.0%) and 5 (12.8%) patients, respectively, and median 2 cycles (range 1-10) of pembrolizumab were given. In 36 patients whose response was assessable, partial response (PR) and stable disease were achieved in 4 (11.1%) and 13 (36.1%), respectively. In 19 (52.8%) patients, progressive disease was the best response. In patients with PR, the median time to response was 2.1 months (95% confidence interval (CI), 0.4 – 3.9). With a median follow-up duration of 4.4 months (95% CI, 2.4 – 6.4), median progression-free survival and overall survival was 1.5 months (95% CI, 0.4 – 2.6) and 4.3 months (95% CI, 2.6 – 6.1), respectively. No grade 3/4 adverse events (AEs) were reported and grade 1/2 fatigue (n = 4, 10.3%) was the most common AE. Conclusions: In PD-L1 positive BTC, pembrolizumab showed modest efficacy with 11.1% of response rates although our patients were heavily pretreated. Considering the limited therapeutic options and poor survival for these patients, further evaluation of immunotherapy including biomarker analysis is needed.