<p>Virus-like particle (VLP) holds great promise for applications in vaccines and tumor immunotherapy. However, their clinical translation has been limited by a lack of comprehensive in vivo studies on immune responses and antigenic toxicity. In this study, we systematically evaluated the efficacy and safety of VLP as immunological agents. We administered Simian Virus 40 (SV40) VLP through subcutaneous injection and analyzed their effects on immune cell populations in key organs. In vivo imaging of mice demonstrated the migration of SV40 VLP between lymph nodes. Flow cytometry revealed that SV40 VLP significantly increased the numbers of CD4⁺ T cells and NK cells in the spleen, along with elevated levels of CD4⁺ T cells in mesenteric lymph nodes. Moreover, SV40 VLP did not significantly affect immune cell populations in the lungs, liver, or kidneys, nor did they alter blood biochemistry or coagulation parameters. Although SV40 VLP alone did not exhibit tumor-treating effects, in vitro imaging suggest that SV40 VLP can target tumor tissues and and quantitative analysis showed SV40 VLP significantly increased TNF-α expression in spleen. These findings suggest that SV40 VLP represent a promising tumor immunotherapy vector with potential for further modification.</p> Graphical Abstract <p></p>