Abstract Mitochondria contain a unique DNA genome (mtDNA) that encodes a number of key components of the respiratory chain. Levels of mtDNA gene expression correlate with the metabolic requirements of mammalian cells. Recently, the role of mitochondrial function for the development and progression of cancer has been highlighted. Oncogenes have been identified to redirect mitochondrial functions to increase cancer cell survival. Increased levels of oxidative phosphorylation have also been implicated in cancer cell resistance towards drugs. In agreement with this notion, compounds that inhibit respiration may specifically target such resistant cells. Finally, it was demonstrated that inhibition of mitochondrial gene expression leads to apoptosis in rapidly dividing cells. Based on these observations, we present a completely novel principle different from known electron transfer chain blockers to treat cancer by inhibiting mtDNA transcription. To this end a study to identify and characterize inhibitors of mitochondrial transcription (IMTs) with lead-like properties was initiated. A high throughput biochemical screening campaign was performed identifying small molecular weight inhibitors of PolRMT, the mitochondrial RNA polymerase, which controls mtDNA transcription. Upon hit optimization, potent compounds with biochemical and cellular IC50s in the low nanomolar range have been identified. IMTs displayed good physicochemical and pharmacological properties. They have been shown to be inactive on other RNA polymerases proving their excellent specificity. Testing IMTs in assays for cancer cell proliferation revealed potent inhibition of sensitive cell lines (double digit nanomolar range). Interestingly, none of the tested primary non-cancer cell lines was affected by the IMTs, indicating a good therapeutic window. Testing selected IMTs in pharmacokinetic studies in vivo revealed high exposures and no detectable adverse effects (single and repeated dosing). Medicinal chemistry optimization will help to identify candidate compounds for efficacy testing in xenograft models. Citation Format: Tim Bergbrede, Axel Choidas, Raffaela Di Lucrezia, Sascha Menninger, Anke Unger, Koch Uwe, Peter Nussbaumer, Nina Bonekamp, Emily Hoberg, Victor Posse, Andrea Felser, Maria Falkenberg, Nils Göran Larsson, Claes M. Gustafsson, Bert M. Klebl. Inhibitors of mitochondrial transcription (IMT) specifically affect cancer cell proliferation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 222.