Self-DNA is present in the cytosol of many cancer cells and can promote effective immune rejection of tumor cells, but the mechanisms leading to the presence of cytosolic DNA are unknown. Here, we report that the cleavage of genomic DNA by DNA structure-specific endonuclease MUS81 and PARP-dependent DNA repair pathways leads to the accumulation of cytosolic DNA in prostate cancer cells. The number of nuclear MUS81 foci and the amount of cytosolic dsDNA increased in tandem from hyperplasia to clinical stage II prostate cancers and decreased at stage III. Cytosolic DNA generated by MUS81 stimulated DNA sensor STING-dependent type I interferon (IFN) expression and promoted phagocytic and T cell responses, resulting in type I and II IFN-mediated rejection of prostate tumor cells via mechanisms that partly depended on macrophages. Our results demonstrate that the tumor suppressor MUS81 alerts the immune system to the presence of transformed host cells. •Presence of cytosolic DNA in prostate cancer cells depends on Mus81•Mus81 induces STING-dependent type I interferon expression•Immune rejection of prostate cancer cells relies on Mus81 and STING•Mus81 enhances innate and adaptive anti-cancer immune responses The mechanisms leading to the presence of cytosolic DNA in cancer cells are unknown. Gasser and colleagues find that the DNA structure-specific endonuclease Mus81 promotes the shedding of genomic DNA into the cytosol of prostate cancer cells. Mus81 stimulates STING-dependent DNA sensor pathways and immune rejection of prostate cancer cells.