BACKGROUND: Human papillomavirus (HPV), particularly high-risk types such as HPV16, is associated with several malignancies, including cervical cancer. Existing therapeutic vaccines targeting HPV oncoproteins E6 and E7 show limited immunogenicity and high production costs. Self-amplifying mRNA (saRNA) vaccines offer a promising alternative by enabling robust antigen expression at low doses. This study evaluated the immunogenicity and antitumor efficacy of a novel saRNA vaccine, JJ-saRNA-HPV01, targeting HPV16 E6/E7 in a preclinical mouse model. METHODS: JJ-saRNA-HPV01, encoding an HPV16 E6-linker-E7 fusion protein, was encapsulated in lipid nanoparticles (LNP). Physicochemical properties (size, PDI, encapsulation efficiency, and zeta potential) were characterized, and in vitro expression was confirmed in 293 T cells by Western blot. Female C57BL/6 mice were immunized intramuscularly with single or double doses (0.1-5 μg). Immune responses were assessed by IFN-γ ELISpot, CD8⁺CD69⁺ T-cell activation, and tumor-infiltrating lymphocyte analysis. Antitumor efficacy was evaluated in TC-1 tumor-bearing mice, with prophylactic and long-term protection tested by tumor challenge and re-challenge. Statistical tests included one-way and two-way ANOVA with multiple comparisons and Kaplan-Meier survival analysis with Mantel-Cox test. RESULTS: JJ-saRNA-HPV01 elicited potent, dose-dependent, E7-specific CD8⁺ T-cell responses in the mouse model. In therapeutic TC-1 models, both 0.1 and 1 µg doses markedly inhibited tumor growth (TGI = 93.0 and 95.7%) and improved survival (p < 0.001), with the 1 µg dose achieving complete regression and 91% survival. Prophylactic vaccination provided 100% protection and cured mice rejected tumor re-challenge, confirming durable E7-specific memory. Mechanistically, vaccination increased intratumoral CD8⁺ infiltration with limited CD4⁺ recruitment, elevated IFN-γ⁺ effector T-cell frequencies (p < 0.001), and reduced PD-1 expression on tumor-infiltrati