Cancer vaccines using dendritic cells (DCs) have been shown to induce antitumor immunity and have recently been applied to non-immunogenic cancers, such as pancreatic cancer. In this study, we utilized DCs loaded with heat-treated tumor lysate (HTL-DC) as a vaccine in order to stimulate antitumor immunity in a murine pancreatic cancer model and compared them to DCs loaded with tumor lysate (TL-DC). The poorly immunogenic mouse ductal pancreatic cancer cell line PANC02 with syngeneic mouse strain C57BL/6 was used as a model. Inducible heat shock proteins (HSPs) were significantly increased in HTL (HSP70 and HSP90). Tumor size measurements indicated that HTL-DC induced stronger tumor suppression than unpulsed DC or TL-DC (43% reduction in tumor volume compared to control group). T cell proliferation assay and IFN-gamma ELISPOT assay showed that T cell activation increased in the following order: DC<TL-DC<HTL-DC. Furthermore, repeated HTL-DC vaccinations led to higher expansion of IFN-gamma-secreting T cells. Cytotoxicity assay revealed that HTL-DC were more efficient in priming PANC02-specific T cells. Our study identifies HTL as an effective source of tumor-associated antigens (TAAs) for pulsing DCs, and demonstrates that HTL-DC can generate a stronger and broader T cell response against fatal cancers, such as pancreatic cancer.