Significantly lower endogenous BTG2 expression was observed in human muscle invasive bladder cancers (MIBC) than matched normal and non-muscle invasive bladder cancers (NMIBC). BTG2 expression was inversely correlated with increased expressions of DNMT1 and DNMT3a in the MIBC, but not in the NMIBC, suggesting a potential role of BTG2 expression in muscle invasion of bladder cancer. Over 90% of tumor tissues revealed strong methylation in CpG islands of BTG2 gene as opposed to no methylation in the normal tissues, implying epigenetic regulation of BTG2 expression in bladder carcinogenesis. By employing EJ bladder cancer cells and demethylating agent, decitabine, transcription of BTG2 was proved to be upregulated by inhibiting DNMT1 expression via CpG islands modification. DNMT1 binding to BTG2 gene further regulated BTG2 expression by chromatin remodeling, such as H3K9-dimethylation and H3K4-trimethylation, and Sp1 activation. Induced BTG2 expression significantly reduced EJ cell tumorigenesis and invasiveness along with induction of G2/M arrest. The present results directly prove an important role of BTG2(/) (TIS) (21/) (PC) (3) gene played in progression of bladder cancers and suggest BTG2(/) (TIS) (21/) (PC) (3) as a promising epigenetic target for prevention of muscle invasion in human bladder cancers. This article is protected by copyright. All rights reserved.