Cardiac fibroblast to myofibroblast phenoconversion is a critical step during the development of cardiac fibrosis. Myofibroblasts chronically remodel extracellular matrix that results in myocardial stiffening, cardiac dysfunction and eventually heart failure. Previously we showed that Meox2, a homeobox transcription factor, can inhibit myofibroblast phenoconversion. Here we show that Zeb2, a repressor of Meox2, plays a crucial role during this phenoconversion process. Zeb2 overexpression significantly upregulates the expression of three key myofibroblast markers: α-SMA, SMemb and ED-A fibronectin in primary rat cardiac myofibroblast. We show that Zeb2 is highly expressed in myofibroblast nuclei whereas it is minimally expressed in fibroblast nuclei. Zeb2 overexpression in myofibroblasts results in a less migratory and more contractile mature myofibroblast phenotype. Moreover, Zeb2 overexpression represses Meox2 expression in endothelial cells. Thus, the current study enhances our understanding of the mechanism behind myofibroblast phenoconversion and provides a basis for developing Zeb2-based novel anti-fibrotic drug in the future.