Background <p>mRNA vaccines have emerged as highly promising therapeutic agents in cancer immunotherapy. Nevertheless, the current limited immunogenicity of target antigens and the lack of a robust systemic immune response remain significant challenges for their effective application. This study aimed to evaluate the immunogenicity of the CD73 protein and elucidate the mechanisms by which a CD73 mRNA vaccine mediates antitumor immune effects in murine models.</p> Methods <p>CD73 mRNA was synthesized by in vitro transcription and formulated into lipid nanoparticles (CD73-LNPs) using a microfluidic device. The immunogenicity of CD73 was assessed using ELISPOT assays. The ability of CD73-LNPs to activate antitumor immune responses was evaluated by flow cytometry. Therapeutic efficacy was tested in murine models of melanoma, cervical cancer, ovarian cancer, and prostate cancer. Mechanistic insights were obtained through single-cell transcriptomics sequencing and bulk RNA transcriptomic analysis.</p> Results <p>The CD73 protein exhibited potent immunogenicity. In vitro, CD73-LNPs promoted the maturation and activation of primary dendritic cells. In vivo, CD73-LNPs induced humoral and cellular immune responses, increased the secretion of interferon-gamma and granzyme B, and effectively suppressed tumor growth across multiple tumor models. Mechanistically, CD73-LNPs activated dendritic cells through toll-like receptor 3 (TLR3) signaling, enhanced dendritic cell activation, and upregulated chemokine receptor expression on CD8⁺ T cells, thereby promoting CD8⁺ T cell infiltration into the tumor microenvironment. Safety evaluations revealed no toxic effects on vital organs.</p> Conclusion <p>CD73 mRNA vaccine safely and effectively induces antitumor immunity through TLR3-dependent mechanisms, highlighting their considerable potential for clinical translation.</p> Graphical Abstract <p></p>