Abstract Cholesterol Epoxide Hydrolase (ChEH) selectively catalyses the hydrolysis of 5,6α-epoxy-cholesterol (5,6α-EC) and 5,6β-epoxy-cholesterol (5,6β-EC) into cholestane-3β,5α,6β-triol (CT). We recently reported that the ChEH activity is carried out by the anti-estrogen binding site (AEBS), formed by 3β-hydroxysterol-Δ7-reductase (DHCR7) and 3β-hydroxysterol-Δ8-Δ7 isomerase (D8D7I) (de Medina et al, PNAS, 2010). In the early 70’s, 5,6-ECs were reported to be carcinogenic and mutagenic, but these data were then refuted, and recently we confirmed that 5,6-ECs are stable and non-alkylating substances. Interestingly, it was reported that CT was genotoxic under strong oxidative stress conditions in yeast. This suggested to us that the inhibition of ChEH may protect cells against cytotoxic insults and may contribute to the anti-proliferative and chemopreventive properties of AEBS/ChEH inhibitors such as tamoxifen (Tam), raloxifene or docosahexaenoic acid that accumulated 5,6-ECs at therapeutic doses. These different data suggested a potential role of ChEH activation in cancer. We have explored this hypothesis in the present study. We show here that the activity of ChEH generated an unexpected metabolite from CT in a large panel of cancer cells. By using RP-HPLC and GC-MS, we determined this unknown metabolite to be the oxysterol: 6-oxo-cholestan-3β,5α-diol OCDO). We showed that OCDO stimulated the growth and invasiveness of different tumor cell lines (1.4-fold and 6-fold respectively for MCF7 cells) and the growth and invasiveness of thyroid tumors (TT) or breast cancers (TS/A, MCF7) grafted into mice. Immunohistology of OCDO-treated tumors showed there was an increase in the expression of the proliferation marker Ki67 compared with control tumors. Interestingly, OCDO formation in tumor cells and tumors grafted into mice was strongly inhibited by ChEH inhibitors known to have anti-tumor activity such as Tam, PBPE or Dendrogenin A (DDA), a steroidal alkaloid discovered in our laboratory. The anti-tumor activity of Tam or DDA against breast tumors implanted into mice was completely reversed by co-treatment with OCDO, indicating that inhibition of ChEH and OCDO production contribute to the anti-tumor mechanism of action of these molecules. In conclusion, the present study identifies OCDO as being an oxidative metabolite of CT with an uncharacterized role of tumor promoter. The inhibition of OCDO production contributes to the anti-tumor activity of Tam and Dendrogenin A. Thus, the use of these ChEH inhibitors appears as a relevant strategy to block the production of this tumor promoter that may be produced during oxidative stress conditions and inflammation driven carcinogenesis. Citation Format: Philippe de Medina, Michael R. Paillasse, Maud Voisin, Marc Poirot, Sandrine Silvente-Poirot. 6-oxo-cholestan-3β,5α-diol (OCDO) is a metabolite produced in tumors by cholesterol epoxide hydrolase activity and a tumor promoter: OCDO inhibition contributes to the anti-tumor activity of tamoxifen and dendrogenin A. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1884. doi:10.1158/1538-7445.AM2013-1884