Background: Tumours that are deficient in DNA mismatch repair (dMMR) have high microsatellite instability (MSI-H) are likely to be immunogenic, triggering upregulation of immune checkpoint proteins. New therapeutic options are needed for patients with advanced colorectal cancer whose disease has progressed after 1 or more lines of therapy. We investigated the safety and efficacy of pembrolizumab, a monoclonal antibody to programmed cell death–1 protein (PD-1 in a cohort of colorectal MSI-H/dMMR cancers patients with previously treated with one line of therapy. Methods: Twenty two eligible patients with histologically/cytologically confirmed MSI-H/dMMR metastatic colorectal cancer who experienced failure with one line of prior therapy received pembrolizumab 200 mg every 3 weeks until disease progression or unacceptable toxicity The primary endpoint was objective response rate (ORR) per RECIST v1.1, as assessed by radiologic review and safety. Tumours were classified as MSI-H/dMMR when expression as detected by immunohistochemistry of at least one of four MMR proteins was absent, or when at least two allelic loci size shifts among the five analyzed microsatellite markers were detected by PCR. Results: Of 22 patients enrolled, median (range) age was 62 (39-78) years. Median follow-up was 15.6 months. The ORR was 36.8 % (95% CI, 29.3% to 42.8%). Median progression-free survival was5.3 months (95% CI, 2.9 to 5.9 months) and median overall survival was 21.5 months (95% CI, 12.8 months to not reached).Treatment-related adverse events occurred in 13 patients (59%). -Four patients (18%) had grade 3 to 5 treatment-related adverse events. There were no treatment-related fatal adverse events. Conclusions: Pembrolizumab monotherapy demonstrated durable clinical benefit and manageable safety in patients with metastatic MSI-H/dMMR colorectal cancer who had previously received 1 line of treatment. Further study of pembrolizumab for this group of patients is warranted.