Radiotherapy induced anti-tumor effects depend on both direct tumor cell death caused by radiation and immune activation mediated by cGAS-STING pathway. Metformin (MTF), which could augment the tumoricidal efficiency of radiation, is indicated to be a radiosensitizer by basic research. However, several large prospective clinical trials proved otherwise. In present study, we intend to interrogate the effects of MTF on radiotherapy-induced anti-tumor immune responses and try to explain the inconsistent outcomings of radiotherapy combined with MTF in basic research and clinical practice. To explore the effects of MTF on radiotherapy induced anti-tumor effects, tumor models were established using E0771, B16F10 and LLC cell lines in both immunocompetent and immunodeficient mice. To investigate the composition and function of immune cells in tumor microenvironments, single-cell transcriptome sequencing of CD45+ cells sorted from tumor microenvironments were carried out, and flow cytometry and multiple immunofluorescence analysis were then performed for validation. To reveal the possible mechanisms, tumor cells were subjected to radiotherapy in the presence or absence of MTF in vitro, and RNA-sequencing was then employed followed by subsequent validation with western blotting, real-time qPCR and flow cytometry. We found that systematic administration of MTF could significantly inhibit radiotherapy-induced anti-tumor effects in immunocompetent mouse models. Single cell sequencing of CD45+ cells sorted from tumor microenvironments and further validation showed that administration of MTF dramatically attenuated the infiltration and cytotoxic capacity of CD8+ T cells after radiotherapy. cGAS-STING pathway in tumor cells was required for maximum efficiency of radiotherapy, while MTF curbed cGAS-STING pathway after radiotherapy in a dose-dependent pattern by enhancing autophagy and reducing cytoplasmic mitochondrial DNA accumulation, which contributed to compromised anti-tumor effects. Our findings indicated that MTF could antagonize radiotherapy-mediated anti-tumor effects by inhibiting the activation of cGAS-STING pathway and subsequent immune responses, which may partially explain the unsatisfied outcomes of radiotherapy combined with MTF in clinical practices. Since the anti-tumor effects of radiotherapy rely not only on the tumor-killing efficiency of radiation but also on systematic immune responses, our findings suggest that cautions are needed when MTF is administrated with radiotherapy in clinical practice.