Celastrol (CSL) is a naturally occurring triterpenoid acid that exhibits anticancer activity, and in KU7 and 253JB-V bladder cells, CSL induced apoptosis, inhibited growth, colony formation and migration and CSL decreased bladder tumor growth in vivo . CSL also decreased expression of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 and several Sp-regulated genes/proteins including vascular endothelial growth factor, survivin and cyclin D1 and fibroblast growth factor receptor-3, a potential drug target for bladder cancer therapy, has now been characterized as an Sp-regulated gene downregulated by CSL. The mechanism of Sp downregulation by CSL was cell context-dependent due to activation of proteosome-dependent (KU7) and -independent (253JB-V) pathways. In 253JB-V cells, CSL induced reactive oxygen species (ROS) and inhibitors of ROS blocked CSL-induced growth inhibition and repression of Sp1, Sp3 and Sp4. This response was due to induction of the Sp repressors ZBTB10 and ZBTB4 and downregulation of miR-27a and miR-20a/17-5p, respectively, which regulate expression of these transcriptional repressors. Thus, the anticancer activity of CSL in 253JB-V cells is due to induction of ROS and ROS-mediated induction of Sp repressors (ZBTB4/ZBTB10) through downregulation of miR-27a and miR-20a/17-5p.