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The spindle assembly checkpoint represents a highly conserved surveillance mechanism which safeguards correct chromosome segregation by delaying anaphase onset until all chromosomes are properly bi-oriented on the spindle apparatus. Non-catalytic functions of the mitotic kinase BUB1 (budding uninhibited by benzimidazoles 1) were reported to be essential for spindle assembly checkpoint activation. In contrast, the catalytic function of BUB1 plays a minor role in spindle assembly checkpoint activation but is required for chromosome arm resolution and positioning of the chromosomal passenger complex for resolution of spindle attachment errors. Here, we disclose for the first time the structure and functional characterization of a novel, first-in-class Bub1 kinase inhibitor. Medicinal chemistry efforts resulted in BAY 1816032 featuring high potency, long target residence time and good oral bioavailablity. It inhibits BUB1 enzymatic activity with an IC50 of 7 nanomol/L, shows slow dissociation kinetics resulting in a long target residence time of 87 min, and an excellent selectivity on a panel of 395 kinases. Mechanistically BAY 1816032 abrogated nocodazole-induced Thr-120 phosphorylation of the major BUB1 target protein histone H2A in HeLa cells with an IC50 of 29 nanomol/L, induced lagging chromosomes and mitotic delay. Persistent lagging chromosomes and missegregation were observed upon combination with low concentrations of paclitaxel. Single agent BAY 1816032 inhibited proliferation of various tumor cell lines with a median IC50 of 1.4 micromol/L and demonstrated synergy or additivity with paclitaxel or docetaxel in almost all cell lines evaluated (minimal combination index 0.3). In tumor xenograft studies BAY 1816032 only marginally inhibited tumor growth as single agent upon oral administration, however, upon combination with paclitaxel or docetaxel a strong and statistically significant reduction of tumor size as compared to the respective monotherapy was observed. Intratumoral levels of phospho-Thr120 H2A were found to be strongly reduced, and no hints on drug-drug interactions were found. In line with the good tolerability in xenograft studies, no relevant findings from non-GLP 2 weeks toxicological studies in rat and dog were reported. Our findings validate the innovative concept of interference with mitotic checkpoints and justify clinical proof of concept studies evaluating BUB1 inhibitor BAY 1816032 in combination with taxanes in order to enhance their efficacy and potentially overcome resistance. Citation Format: Gerhard Siemeister, Anne Mengel, Wilhelm Bone, Jens Schroder, Sabine Zitzmann-Kolbe, Hans Briem, Amaury E. Fernandez-Montalvan, Simon Holton, Ursula Monning, Oliver von Ahsen, Sandra Johanssen, Arwed Cleve, Marion Hitchcock, Kirstin Meyer, Franz von Nussbaum, Michael Brands, Dominik Mumberg, Karl Ziegelbauer. BAY 1816032, a novel BUB1 kinase inhibitor with potent antitumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 287. doi:10.1158/1538-7445.AM2017-287