Abstract: Ninety percent of pancreatic ductal adenocarcinoma was induced by KRAS gene mutation in clinic. At present, there was a lack of effective targeted therapies for KRAS mutated cancer. Immunotherapy had emerged as a prominent approach for tumor treatment. This study aimed to develop a KRAS-targeted immunotherapy by enhancing immune response of anti-KRAS mutation in vivo. We constructed four plasmids with several KRAS-mutated gene sequences, HSP70 and GM-CSF. The order and linker of these KRAS-mutated gene sequences were different within these four plasmids, but they all contained the KRAS^Q61H mutation. These plasmids were packaged into adenovirus 5, and their capacity of anti-tumor was analyzed in vitro and in vivo. The results showed that the four plasmids could be well expressed KRAS antigen, HSP70 and GM-CSF in vitro, as well as elicited immune responses in mice. Furthermore, a pancreatic carcinoma cell line with KRAS^Q61H mutation, Pan02-Q61H was subcutaneously injected into mice. Among the four vectors, the Ad-SNP1 was best for inhibited the tumor growth. They also induced immune responses, led to inhibition of tumor growth in a Pan02 cells a transplanted tumor mouse models which contained KRAS^Q61H mutation sites, and there were significant differences in efficacy among the four drugs. We selected the Ad-SNP1 vector with the best pharmacological effect from the four drugs, laying a foundation for further research in the future, and hoping that it can provide new treatment opportunities for pancreatic cancer patients.