The presence of peritoneal metastasis in patients with pancreatic cancer is associated with poor prognosis. Chemotherapy and radiotherapy may result in poor prognosis in patients with pancreatic cancer. However, immunotherapy improves prognosis even at an advanced stage of the disease. The present study reported a case of a combined therapy of autologous ex vivo expanded natural killer (NK) cells and programmed cell death 1 (PD-1) inhibitor in a patient with pancreatic cancer and peritoneal metastasis. The NK cells were expanded ex vivo and intravenously injected. This was followed by intravenous administration of two dosages of PD-1 inhibitor. Computed tomography and magnetic resonance imaging were performed to assess the size of tumor before and after the combined therapy. In addition, the blood sample and ascites were collected and analyzed before and after the combined therapy. Flow cytometry was carried out to measure the subsets of T cells and macrophages in the collected ascites. Meanwhile, the levels of cytokines in the ascites were quantified through enzyme-linked immunosorbent assay, and Luminex assays were conducted on the supernatant. It was revealed that after the combined therapy, cancer cells disappeared in the ascites, and the T cells were activated, which could be confirmed by the decreased levels of PD-1 and T cell immunoglobulin and mucin domain-containing protein 3. Also, the functioning of macrophages was improved, as shown by the increased level of CD86 and the reduced levels of CD206 and HLA-DR. Notably, the levels of cytokines (transforming growth factor-ß, vascular endothelial growth factor, and interleukin-10) in ascites were significantly upregulated after the combined therapy. In conclusion, it was evident that NK cells combined with PD-1 inhibitor improved the immune microenvironment of carcinomatosis in the peritoneal cavity. Therefore, the combined therapy may be beneficial for suppressing pancreatic cancer and the presence of metastases in the peritoneal cavity. However, there is a need for additional randomized studies to confirm the efficacy of combined therapy.