Abstract Background: NAD(P) Dependent Steroid Dehydrogenase-Like (NSDHL) is an enzyme involved in post-squalene cholesterol biosynthesis. We recently suggest new insights into the critical role of the NSDHL involved in breast tumor growth and metastasis and its potential for targeted cancer therapy. Compound 9, which is a novel inhibitor of NSDHL, was kindly provided by Dr. Lee B-J in Seoul National University College of Pharmacy. This study aimed to investigate the anti-cancer potential of Compound 9 in breast cancer. Methods: Therapeutic potential of Compound 9 (10~100 μM) was investigated in several human breast cancer cell lines. Total intracellular cholesterol level was measured using Total Cholesterol Assay Kit. Cell cycle analysis using DNA staining with PI, apoptosis analysis using Annexin V/PI-labeling, and transwell migration assay were performed. The Synergistic anticancer effect of compound-9 and paclitaxel was evaluated by crystal violet assay. Western blot and immunofluorescent staining were assessed for investigating the mechanism by which Compound 9 exerts the anti-cancer effect. Results: Time-dependent intracellular uptake of Compound 9 by breast cancer cells was observed. Compound 9 induced cytotoxic, anti-proliferative, and apoptotic effects and cell cycle arrest (G0/G1), and suppressed migration activity on diverse breast cancer cell lines in a time- and dose-dependent manner. Total intracellular cholesterol level was reduced by Compound 9. Combination with Compound-9 and Paclitaxel led to synergistic cytotoxicity. Compound-9 increased the autophagy-related proteins (P62, LC3II) but decreased the anti-apoptotic proteins (XIAP, BCL2). Conclusion: Our data show that Compound 9 exerts strong anti-proliferative and cytotoxic activity in breast cancer cells, suggesting potential additional utility as an anti-cancer drug. Further studies are necessary to validate the potential anti-cancer effect of Compound-9 in breast tumor mice models. Citation Format: So-Hyun Yoon, Hoe Suk Kim, Moonjou Baek, Seungyeon Ryu, Sangeun Lee, Han-Byoel Lee, Bong-Jin Lee, Wonshik Han. Novel NSDHL inhibitor (Compound 9) exerts anti-proliferative and cytotoxic activity in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1800.