Introduction: Quiescent cardiac fibroblasts are required for tissue homeostasis of the myocardium. Upon activation by various stressors, cardiac fibroblasts exhibit the hyper-secretory, hyper-proliferative myofibroblast phenotype. We have shown that overexpression of the endogenous TGF-β1 repressor, Ski, functions to deactivate myofibroblasts to a fibroblast-like phenotype. In addition, Ski downregulates the expression of Hippo pathway effectors and the expression of pro-fibrotic Zinc finger E-box Binding homeobox 2 (ZEB2). Previous studies have shown that the Hippo signaling pathway regulates ZEB2 expression; thus, we examined the hypothesis that Ski’s downregulation of ZEB2 is mediated by the Hippo signaling pathway.Methods: Rat cardiac fibroblasts were cultured on compressible silicone substrata with an elastic modulus biomimetic to healthy myocardium (E = 5 kPa). Cells were then transduced with adenoviral vectors to overexpress Hippo effectors Yes-Associated Protein (YAP) and Transcriptional co-Activator with PDZ-binding motif (TAZ), as well as anti-fibrotic Ski. Conversely, pro-fibrotic gene expression was targeted using RNAi-mediated gene knockdown. Following treatment, pro-fibrotic gene expression and phenotype were examined.Results: Overexpression of Hippo effectors in cardiac fibroblasts induced the expression of ZEB2 (p=0.0137, n=3) myofibroblast markers such as EDA-containing cellular fibronectin (p=0.0157, n=3) on 5 kPa substrata. Conversely, ectopic expression of nuclear Ski in activated myofibroblasts induced the degradation of endogenous TAZ (p=0.0073, n=3) but not YAP (p=0.34, n=3). ZEB2 knockdown studies suggest that this transcription factor is not essential for myofibroblast activation; thus it is likely that increased ZEB2 expression during cardiac remodelling is not a cause, but a result of fibrogenesis.Conclusions: Our data suggest that Ski’s effects on ZEB2 may be mediated via the Hippo signalling pathway, which postulates a novel mechanism by which Ski promotes the fibroblast phenotype. The Ski-Hippo-ZEB2 signalling axis may provide a relatively selective therapeutic target in the treatment of cardiac fibrosis.