Epigenetic programming within the cardiac stem cell (CSC) niche controls CSC specification and differentiation, but the identity of putative mediators is poorly characterized. To dissect these mechanisms, we used primary and clonally expanded Sca-1+/c-kit+ CSC from murine adult hearts and stimulated cardiomyocyte differentiation upon culture in a differentiation medium (DIFF) containing 5'-Azacytidine and transforming growth factor β1 or co-cultured with rat cardiomyocytes. With this model, we previously showed that inducible deletion of β-catenin enhanced CSC differentiation in vitro and in vivo. Accordingly, we detected a constitutive activity of Wnt/b-catenin pathway in undifferentiated CSC and a downregulation of Wnt target genes (in % of ctl: Axin2: 36±4%; Snai2: 46±7%; n=3; p<0.01) in CSC treated with DIFF medium, associated with an upregulation of Wnt antagonist, Wif-1 (310±57%; n=3, p<0.05). Like several Wnt/b-catenin repressor genes, Wif-1 gene expression is DNA methylation sensitive and susceptible to be regulated by the de novo DNA methyltransferases Dnmt3. Indeed, Dnmt3a was downregulated in CSC treated with DIFF, while siRNA targeting Dnmt3a upregulated Wif-1 expression in non-differentiated cells (425±254%; n=4, p<0.05) and promoted CSC cardiac differentiation in co-culture assay (assessed by quantitative expression of cTnT; 162±7% vs. siRNA-scramble; n=7, p<0.001). In parallel, we found an early upregulation of miR-29a (444±43%; n=3, p<0.01), a well-known regulator of Dnmt3a, in DIFF-treated CSC. Indeed, early treatment of CSC with miR-29a mimic decreased Dnmt3a protein level and increased Wif-1 expression (839±257%; n=4, p<0.05). Reciprocally, CSC treatment with anti-miR-29a (LNA-miR-29a; vs scramble ctl) upregulated Dnmt3a and downregulated Wif-1 expression (51±9%; n=7, p<0.01). Importantly, this treatment also significantly decreased CSC differentiation in co-culture assay (63±4% vs. LNA-scramble; n=11, p<0.001). Altogether, this suggests that upregulation of miR-29a promotes differentiation (and blockade of miR-29a inhibits differentiation) through Dnmt3a-dependent regulation of the Wnt inhibitor, Wif-1. In support of this, co-treatment with anti-miR-29a and siRNA-Dnmt3a abrogates the above effect both on Wif-1 expression and CSC differentiation (compared to treatment with respective scramble, n=7). We conclude that differentiation of CSC involves the epigenetic regulation of canonical Wnt/β-catenin activity through miR-29a and Dnmt3a-dependent control of expression of the endogenous inhibitor, Wif-1, possibly amenable to therapeutic modulation for cardiac repair.