Mesenchymal stem cells (MSCs) specifically differentiate into cardiomyocytes as a potential way to reverse myocardial injury diseases, and uncovering this differentiation mechanism is immensely important. We have previously shown that histone acetylation/methylation and DNA methylation are involved in MSC differentiation into cardiomyocytes induced by islet-1. These modifications regulate cardiac-specific genes by interacting with each other in the promoter regions of these genes, but the molecular mechanism of these interactions remains unknown. In this study, we found that the key enzymes that regulate GATA4/Nkx2.5 expression are Gcn5/HDAC1, G9A, and DNMT-1. When α-methylene-γ-butyrolactone 3 (MB-3) was used to inhibit Gcn5 expression, we observed that the interactions among these key enzymes in the GATA4/Nkx2.5 promoters were blocked, and MSCs could not be induced into cardiomyocytes. Our results indicated that islet-1 could induce Gcn5 binding to GATA4/Nkx2.5 promoter regions and induce the interactions among Gcn5, HDAC1, G9A and DNMT-1, which upregulated GATA4/Nkx2.5 expression and promoted MSC differentiation into cardiomyocytes.