Tumor-derived total RNA (TdRNA) and cell lysate (TCL), with almost all the relevant tumor antigens, represent attractive alternative sources of antigens in antitumor immunotherapy. However, the comparison of their capacity to elicit immune responses against breast cancer is still lacking. In this study, the antitumor immune effects of TdRNA and TCL were systematically compared. We isolated TdRNA and TCL from 4T1 mouse breast cancer cells, and found that both sources of antigens could stimulate the maturation of dendritic cells (DCs) at the cellular and in vivo levels, and induce robust cellular immune responses, as evidenced by the increased percentages of both CD4⁺ and CD8⁺ T cells in the inguinal lymph nodes and spleen. But TdRNA performed stronger immunoactivities than TCL on the increase of T cell population through DCs activation. Additionally, the synergistic antitumor efficacy of paclitaxel (PTX) with TdRNA and TCL respectively was further evaluated in the murine 4T1 tumor model. Compared with TCL, TdRNA could inhibit tumor growth more effectively with low systemic toxicity when combined with PTX, which was, at least in part, attributable to the improvement of systemic immune function and tumor immune infiltration. Overall, TdRNA outperforms TCL in antitumor immunity, and is expected to be a promising candidate for application as the source of tumor antigens.