The repair of DNA double-stranded breaks (DSB) is crucial for genomic stability in both normal and cancer cells. In cancer cells suffering from both intrinsic stresses and extrinsic DNA damage induced by radiation or chemotherapy, efficient DSB repair is essential for cell survival. Therefore, understanding the mechanisms of DSB repair is critical for exploiting the genomic vulnerabilities of cancer cells. Recent studies by us and others revealed that the DSB in transcribed regions of the genome are repaired through a novel mRNA dependent DNA repair (RDDR) mechanism. Our goal is to target the RDDR pathway in cancer therapy. We identified a specific mRNA methyltransferase, TRDMT1, which is a writer of mRNA methyl-5-cytosine at DNA damage sites and an initiator of RDDR. Using multiple live-cell and biochemical platforms that we developed, we identified lead compounds that specifically inhibit TRDMT1. In breast and ovarian cancers, Homologous recombination (HR)-deficient tumors and over half of HR-proficient tumors may harbor increased mRNA-dependent repair activity, presenting an attractive opportunity for TRDMT1i therapy. Our study may establish the first approach to target this pathway in cancer therapy and broaden the exploitation of genomic instability in both HR-deficient and – proficient tumors.