SOX9 is a key transcription factor during cell differentiation, sex determination, and tumorigenesis. However, the detailed mechanisms of its targeting strategy remain elusive. To investigate possibilities of targeting SOX9 with epigenetic drugs and the precise underlying mechanisms, two human cancer cell lines were chosen as model systems, which showed high SOX9 expression and anti-tumorigenic effects upon loss of SOX9. Histone acetylation-related screening of a small panel of epigenetic drugs revealed that the bromodomain reader inhibitor JQ1 dramatically downregulated SOX9 through multiple regulation steps, namely, transcription, BRD4-SOX9 protein-protein interaction, and further protein stability. These findings suggest that BRD4 inhibition is a novel therapeutic strategy for diseases characterized by SOX9 overexpression. (C) 2019 Elsevier Inc. All rights reserved.