Dendritic cell (DC) based anti-cancer immunotherapy is believed to be a promising treatment. However, appropriate conditioning including the method of antigen loading and stimulation for DC maturation is still unclear. Total RNA pulsing is one of the most attractive methods to load antigen, since RNA is easily replicated by the PCR technique and is absolutely free of tumor cell contamination. On the other hand, CD40 ligation is capable of producing one of the most potent signals for immature DCs to start functional maturation, which is required to induce adaptive immunity, resulting in altered migration ability to secondary lymphoid organs and augmented antigen presenting activity. Here, we demonstrate that DCs pulsed with total RNA extracted from tumor cells required CD40 stimulation with an appropriate sequence to present tumor-associated antigens. RNA derived antigens were presented for both CD4+ and CD8+ T cells in an antigen-specific manner. Dendritic cells that were pulsed with RNA followed by the stimulation through CD40 successfully primed antitumor effector T cells in draining LNs and subsequently induced antitumor protective immunity.