The therapeutic potential of cancer immunotherapy has been increasingly recognized, particularly in strategies that exploit the body’s own immune system to target and eliminate tumor cells. One promising approach involves the use of dendritic cells (DCs) as powerful antigen-presenting cells to generate antigen-specific T cells capable of targeting cancer cells. In this study, we explore the use of placental gp96, a stress-induced protein overexpressed in various tumors, to prime expanded dendritic cells for the generation of antigen-specific T cells. The expanded DCs, loaded with placental gp96, were shown to induce potent anti-tumor immunity in vitro, as evidenced by the strong T cell proliferation, activation, and cytotoxicity against cancer cell lines such as MCF-7, glioblastoma cell line U87MG and the neuroblastoma cell line SH-SY5Y. Notably, placental gp96-loaded DCs induced significantly higher cytotoxicity (67-71%) against tumor cell lines compared to recombinant gp96 (23-26%, p < 0.001), underscoring its clinical potential. This activity may be mediated by various tumor-associated peptides presented on the placental gp96. Moreover, this anti-tumor effect is MHC-restricted. These findings suggest that placental gp96-loaded expanded DCs hold significant promise as a novel immunotherapy strategy for cancers.