A zinc finger protein, GATA4, is one of the hypertrophy-responsive transcription factors and increases its DNA binding and transcriptional activities in response to hypertrophic stimuli in cardiac myocytes. Activation of GATA4 during this process is mediated, in part, through acetylation by intrinsic histone acetyltransferases such as a transcriptional coactivator p300. However, p300-targeted acetylated sites of GATA4 during myocardial cell hypertrophy have not been identified. By mutational analysis, we showed that 4 lysine residues located between amino acids 311 and 322 are required for synergistic activation of atrial natriuretic factor and endothelin-1 promoters by GATA4 and p300. A tetra-mutant GATA4, in which these 4 lysine residues were simultaneously mutated, retained the ability to localize in nuclei and to interact with cofactors including FOG-2, GATA6, and p300 but lacked p300-induced acetylation, DNA binding, and transcriptional activities. Furthermore, coexpression of the tetra-mutant GATA4 with wild-type GATA4 impaired the p300-induced acetylation, DNA binding, and transcriptional activities of the wild type. When we expressed the tetra-mutant GATA4 in neonatal rat cardiac myocytes using a lentivirus vector, this mutant suppressed phenylephrine-induced increases in cell size, protein synthesis, and expression of hypertrophy-responsive genes. However, its expression did not affect the basal state. Thus, we have identified the most critical lysine residues acting as p300-mediated acetylation targets in GATA4 during hypertrophic responses in cardiac myocytes. The results also demonstrate that GATA4 with simultaneous mutation of these sites specifically suppresses hypertrophic responses as a dominant-negative form, providing further evidence for the acetylation of GATA4 as one of critical nuclear events in myocardial cell hypertrophy.