Hollow organs exposed to pathologic stimuli undergo phenotypic modulation characterized by altered expression of smooth muscle contractile proteins and loss of normal function. The molecular mechanisms that regulate smooth muscle differentiation, especially in organs other than the vasculature, are poorly understood. In this study, we describe a role for the GATA-6 transcription factor in regulation of human bladder smooth muscle differentiation. Knockdown of endogenous GATA-6 in primary human bladder smooth muscle cells (pBSMC) led to decreased mRNA levels of the differentiation markers alpha-smooth muscle actin (-SMA), calponin and smooth muscle myosin heavy chain (SM-MHC). Similar effects were obtained following down-regulation of GATA-6 by forskolin-induced elevation of intracellular cyclic AMP levels. Forskolin treatment of pBSMC abolished recruitment of GATA-6 to the -SMA promoter in vivo and reduced activity of human -SMA promoter-directed gene expression by >60%. This inhibitory effect was rescued by enforced expression of wild type GATA-6, but not by a zinc-finger deleted mutant, GATA-6-ZF, which lacks DNA-binding ability. In silico analysis of a region of the human -SMA promoter described previously as a transcriptional enhancer identified a putative GATA-binding site at position -919/-913. Point mutation of this site in the -SMA reporter construct abrogated GATA-6-induced activation of promoter activity. Together, these results provide the first evidence for a functional role for GATA-6 in regulation of bladder smooth muscle differentiation. In addition, these findings demonstrate that GATA-6 regulates human -SMA expression via a novel regulatory cis element in the -SMA promoter-enhancer.