Single-agent immune checkpoint blockade has shown no clinical benefits in pancreatic cancer. Recently, the programmed cell death protein 1 (PD-1) antibody pembrolizumab has been recommended as a treatment option for high tumor mutational burden (TMB) solid tumors based on the data from a basket trial. However, no pancreatic cancer patients were enrolled in that trial. Whether pancreatic cancer patients with high TMB respond to PD-1 blockade as well remains unclear. Here, we report a case with a partial response to single-agent immunotherapy with pembrolizumab in pancreatic cancer with high TMB after the failure of several lines of chemotherapy. This result indicates that single-agent immunotherapy may be effective in pancreatic cancer patients with high TMB. In addition, in order to understand the basic immune state of our patients, we also analyzed the changes in immune cells in peripheral blood with cytometry by time-of-flight mass spectrometry (CyTOF) before and after pembrolizumab treatment.