It is unknown whether the route of administration impacts dendritic cell (DC)-based immunotherapy for pancreatic ductal adenocarcinoma (PDAC). We compared the effect of intraperitoneal (ip), subcutaneous (sc), and intratumoral (it) administration of DC vaccine on induction of antitumor responses in a KPC mouse model of PDAC. Histological analysis and flow cytometry were used to evaluate tumor progression and antitumor immunity after different routes of DC vaccination. Using a flank mouse model of PDAC, we found that the it route of DC vaccination had no significant effect on tumor growth rates compared with ip and sc routes (ip 6.66±2.58% vs sc 6.79±1.36% vs it 8.57±2.36%; P= 0.33). However, in an orthotopic PDAC model, ip injection of DC vaccine effectively suppressed tumor growth, inhibited tumor progression, and increased antitumor immunity compared with sc vaccination (tumor weight: ip 71.60±15.55 mg vs control 200.40±53.04 mg; P= 0.048; sc 151.40±41.64 mg vs control 200.40±53.04 mg; P= 0.49). Our study suggests that immunization via an ip route results in superior antitumor immune response and tumor suppression when compared with other routes.