<p>Oncolytic virus M1 is a promising anticancer agent; however, its therapeutic efficacy is often limited by insufficient intratumoral viral replication and host antiviral immunity. Sirolimus, an mTOR inhibitor widely used in transplantation immunosuppression, has demonstrated potential to modulate antiviral responses. This study investigates whether sirolimus potentiates the efficacy of M1 virotherapy and elucidates the underlying mechanisms. Sirolimus significantly enhanced the antitumor efficacy of M1 virus in murine prostate cancer and liver cancer models, leading to reduced tumor growth. This synergistic effect remained evident in CD8⁺ T cell-depleted mice, indicating that the therapeutic benefit is independent of adaptive cytotoxic immunity. Mechanistically, sirolimus markedly increased M1 viral replication in tumor tissues, accompanied by enhanced tumor cell-cycle arrest and apoptosis. Notably, sirolimus selectively amplified viral load within tumors but not in normal organs, demonstrating tumor-specific viral enrichment and safety. Further analyses revealed that this increase in intratumoral virus was driven by mTOR pathway inhibition rather than alterations in macrophage or NK cell populations. Transcriptomic profiling and molecular validation indicated that sirolimus-mediated mTOR suppression downregulated key type I interferon–stimulated genes (<i>Ifitm1, Stat1, Ifit3</i>), thereby attenuating intrinsic antiviral defenses and facilitating viral amplification. In summary, sirolimus potentiates M1 oncolytic virotherapy by selectively enhancing viral replication in tumors via mTOR inhibition and suppression of type I interferon signaling, independent of CD8⁺ T cell-mediated immunity. These findings establish a mechanistic rationale for combining mTOR inhibitors with oncolytic viruses to achieve dual benefits of enhanced viral oncolysis and controlled immunosuppression, with translational relevance for cancer patients requiring long-term immunosuppressive therapy.</p><p></p>