GATA4 is a particularly important cardiogenic transcription factor and serves as a potent driver of cardiogenesis. Recent progress in the field has made it clear that histone acetylation can influence gene expression through changing the structure of chromatin. Our previous research had revealed that hypo-acetylation could repress gata4 expression in cardiocytes, however the underlying mechanism by which this occurred was still unclear. To reveal the mechanism of histone acetylation involved in the regulation of gata4 transcription, we concentrated on P300, one of the important histone acetyltransferase associated with cardiogenesis. We found that P300 participated in gata4 expression through regulating histone acetylation in embryonic mouse hearts. RNAi-mediated downregulation of P300 modulated the global acetylation of H3 and the acetylation of H3K4, H3K9, and H3K27 in gata4 and Tbx5 promoters. Interestingly, there was an obvious inhibition of gata4 transcription, whereas Tbx5 was not influenced. Furthermore, SGC-CBP30, the selective inhibitor of the bromodomain in CBP/P300, downregulated gata4 transcription by repressing the acetylation of H3K4, H3K9, and H3K27 in the gata4 promoters. Taken together, our results identified that acetylation of H3K4, H3K9, and H3K27 mediated by P300 plays an important role in regulation of gata4 expression in cardiogenesis.