Background: This study investigated the administration of combination therapy, allogeneic natural killer (NK) cells and pembrolizumab, in the treatment of advanced biliary tract cancer to determine the safety and tolerability (phase 1), and the efficacy and safety (phase 2a). Methods: Forty patients (phase 1, n=6; phase 2a, n=34) were enrolled between December 2019 and June 2021. The patients were inoperable and no further conventional chemotherapy was anticipated after finishing at least one chemotherapy. The patients received 3x10⁶ NK cells/kg of highly activated allogeneic NK cells (“SMT-NK”) on weeks 1 and 2 and 200 mg of pembrolizumab (Keytruda) on week 1. No treatment was given in week 3. This 3-week schedule (1 cycle) was repeated until confirmed disease progression, intolerable adverse events (AEs), patient withdrawal of consent, or finishing the maximum treatment schedule. The tumor response was evaluated after every three cycles. Results: In phase 1, 4 patients (66.7%) experienced 7 AEs, but no severe AEs directly related to the combination of the two drugs was observed. In phase 2a, 126 AEs occurred in 29 patients (85.3%). Severe AEs (≥ grade 3) were reported in 16 patients (47.1%). No dose limiting toxicity was reported. The overall response rate (ORR) was 17.4% in the full-analysis set and 50.0% in the per-protocol set. Conclusions: SMT-NKs plus pembrolizumab resulted in no severe AEs directly related to the drug combination. The combination therapy also exerted antitumor activity with improved efficacy compared to recent monotherapy with pembrolizumab in patients with advanced biliary tract cancer. A multi-center, randomized, placebo-controlled, open-label, phase 2b clinical trials to evaluate the antitumor activity of combination therapy of SMT-NKs and pembrolizumab versus pembrolizumab monotherapy in patients with advanced biliary tract cancer is now ongoing with the aim of enrolling 128 patients (ClinicalTrials.gov identifier: NCT05429697). To date, 38 patients have been enrolled and randomly assigned to each group. Within the median follow up period of 5.3 months, the disease control rate (DCR, complete response + partial response) was 27.2% in the combination therapy of SMT-NKs and pembrolizumab group, and 7.1% in the pembrolizumab monotherapy group. The ORR was 54.5% in the combination therapy group and 42.9% in the monotherapy group. So far, no severe drug-related AEs was reported. Clinical trial information: NCT03937895.