Abstract Forkhead box M1 (FOXM1) is a transcription factor of considerable importance. Aberrant overabundance of FOXM1 through mutations in upstream regulators or gene amplification is now known to be a driving factor of most human cancers. Further, FOXM1 has prognostic value as expression correlates with severity of disease. Thus, chemical inhibition of FOXM1 has become a major goal. To address this need, we designed a novel in vitro assay to detect disruption of FOXM1 DNA binding. We executed a screen of 400,000 compounds from the NIH Molecular Library Small Molecule Repository, consisting of diverse drug-like molecules intended as starting points for medicinal chemistry lead development. After iterative and orthogonal counter screens, we ultimately identified the small molecule FDI-6 as a potent inhibitor of FOXM1. We characterized the perturbation in detail by biophysical analyses and confirmed that FDI-6 binds FOXM1 protein directly. The molecule was cytotoxic to multiple cancer cell lines (GI50 ≈ 10μM) and we demonstrated that the inhibitor displaces FOXM1 protein from promoters of target genes (AURKB, CCNB1, CDC25B) using an MCF-7 breast cancer model. To generalize the effect, we used chromatin immunoprecipitation and next generation sequencing (ChIPseq) to show that the inhibitor physically displaces FOXM1 from consensus binding motifs across the entire genome, reducing FOXM1 peaks by an average of over 60%. Transcriptome-wide expression profiling by RNAseq further confirmed that this displacement by FDI-6 selectively down-regulates the global FOXM1 transcriptional program, suppressing mitotic entry and cell-cycle progression. Importantly, we found that FDI-6 is specific for FOXM1 and has no effect on the expression of genes regulated by related forkhead family factors, which exhibit homology with the DNA binding domain of FOXM1. We are now evaluating the efficacy of this compound in allograft mouse models of FOXM1-driven breast cancer. Our study shows that the genomic interaction of this clinically important transcription factor can now be manipulated with small molecules to regulate the expression of key gene families. This improves our ability to probe transcription factor function, helps establish the oncogenic roles in different disease contexts and demonstrates clear potential for FOXM1 to be pursued as a clinical target in the future. Citation Format: Michael Gormally, Giovanni Marsico, Ganesha Rai, Christopher Lowe, Craig Thomas, David Maloney, Sam Michael, Dijana Matak-Vincovic, Ajit Jadhav, Anton Simeonov, Shankar Balasubramanian. Transcription factor as target: Novel small molecule inhibits FOXM1 DNA binding and oncogenic gene products. [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 3088.