Pancreatic cancer is an extremely aggressive malignancy with a dismal prognosis. Cancer patients and tumor-bearing mice have multiple immunoregulatory subsets including regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSC) that may limit the effectiveness of anti-tumor immunotherapies for pancreatic cancer. It is possible that modulating these subsets will enhance anti-tumor immunity. The goal of this study was to explore depletion of immunoregulatory cells to enhance dendritic cell (DC)-based cancer immunotherapy in a murine model of pancreatic cancer. Flow cytometry results showed an increase in both Tregs and MDSC in untreated pancreatic cancer–bearing mice compared with control. Elimination of Tregs alone or in combination with DC-based vaccination had no effect on pancreatic tumor growth or survival. Gemcitabine (Gem) is a chemotherapeutic drug routinely used for the treatment for pancreatic cancer patients. Treatment with Gem led to a significant decrease in MDSC percentages in the spleens of tumor-bearing mice, but did not enhance overall survival. However, combination therapy with DC vaccination followed by Gem treatment led to a significant delay in tumor growth and improved survival in pancreatic cancer–bearing mice. Increased MDSC were measured in the peripheral blood of patients with pancreatic cancer. Treatment with Gem also led to a decrease of this population in pancreatic cancer patients, suggesting that combination therapy with DC-based cancer vaccination and Gem may lead to improved treatments for patients with pancreatic cancer.