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Personalized neoantigen mRNA vaccine showed high potency to treat advanced melanoma and pancreatic cancer in recent clinical trials. Strategies to further increase its therapeutic efficacy are highly demanded. This study explored flagellin to increase model antigen ovalbumin (OVA) mRNA-induced cytotoxic T lymphocyte (CTL) responses and anti-tumor immunity in OVA-expressing B16F10 melanoma models. To minimize the potential negative impact of flagellin-induced signaling pathways on OVA mRNA translation, flagellin mRNA was used in our studies. We found flagellin-OVA mRNA (co-expression) but not flagellin mRNA/OVA mRNA (separate expression) significantly increased granzyme B, perforin, and interferon γ-secreting CD8⁺ and CD4⁺ T cell levels in spleen of tumor-bearing mice. Flagellin co-expression but not separate expression also significantly increased perforin⁺ CD8⁺ tumor-infiltrating lymphocytes (TILs) and perforin⁺ and granzyme B⁺ CD4⁺ TILs. To our surprise, flagellin co-expression and separate expression significantly reduced CD8⁺ TILs as compared to OVA mRNA alone. The ratio of CD8⁺ to CD4⁺ TILs was significantly increased by OVA mRNA vaccination alone or with flagellin co-expression but not with flagellin separate expression. The ratio of CD8⁺ to CD4⁺ TILs was significantly higher in flagellin co-expression than separate expression group. Collectively, flagellin co-expression more significantly reduced tumor growth rate than flagellin separate expression but only slightly reduced tumor growth rate as compared to OVA mRNA alone. In summary, these results support flagellin co-expression to enhance mRNA vaccine-induced CTL responses, yet strategies are demanded to promote tumor infiltration of elicited peripheral CTLs.