Background: Recently, we reported that histone deacetylase (HDAC) inhibitors block cardiac hypertrophy and that activation of HDAC2, one of the class I HDACs, is required for hypertrophy to develop. In the present study, we used rat neonatal cardiomyocytes and H9c2 cells to find the downstream target of HDAC inhibitor. Methods & Results: We used trichostatin A (TSA) as an inhibitor of both class I and II HDACs and SK7041 (SK) as a class I-selective blocker. Both TSA and SK attenuated the expression level and promoter activity of Nppa (natriuretic polypeptide precursor type A) and Myh7 (myosin heavy polypeptide 7), which are fetal genes associated with hypertrophy. Furthermore, this inhibition was specific to muscle cells. Promoter mapping analysis revealed that the Nppa promoter region from -130 to approximately -105, which contains binding sites for Krüppel-like factor 4 (KLF4), is responsible for the HDAC inhibitor-mediated inhibition. SK-induced repression of Nppa promoter activity was attenuated when KLF4-binding element was deleted or disrupted. Klf4 was upregulated by HDAC inhibitors, whereas it was down-regulated by phenylephrine in cardiomyocytes or by partial aortic constriction in mice. Klf4 successfully recruited the proximal Nppa promoter region flanking the KLF4-binding element in cardiomyocytes and the recruitment was reduced by treatment with phenylephrine. Overexpression of Klf4 blocked the agonist-induced increase in cardiomyocyte size, stress fiber, [³H]-leucine incorporation, and Nppa promoter activity as well as Nppa transcript level. However, promoter activity was not prominently inhibited when the KLF4-binding element was disrupted or when a small inhibitory RNA to KLF4 was transfected into cells. Knocking down of KLF4 enhanced hypertrophic phenotypes. Conclusions: These results suggest that KLF4, a novel anti-hypertrophic transcriptional regulator, mediates the HDAC inhibitor-induced prevention of cardiac hypertrophy.