Background: It has been demonstrated that orphan nuclear receptor, small heterodimer partner (SHP; NR0B2) directly modulates the activities of conventional nuclear receptors and regulates a variety of cellular events such as cell proliferation, differentiation and metabolism in liver and bone. However, the role of SHP in heart has not yet been elucidated. Thus, in this study, we tried to investigate the functional roles of SHP in heart and cardiac hypertrophy. Methods and Results: We observed that SHP was expressed in rat heart. In rat neonatal rat cardiomyocytes model, cardiomyocyte hypertrophy was induced by phenylephrine (PE), 慣-adrenergic agonist, and SHP was down-regulated by phenylephrine (PE). Transient transfection of SHP decreased the promoter activity of Nppa (natriuretic polypeptide precursor type A) a cardiac hypertrophy marker, in dose-dependent manner. Adenovirus-mediated overexpression of SHP (Ad-SHP) blocked cardiac specific gene expression such as GATA4 and serum response factor (SRF). The increase in [3H]-leucine incorporation induced by PE, endothelin-1 (ET-1), or fetal bovine serum (FBS) was dramatically reduced by infection of Ad-SHP. Likewise, increases in cell size with those hypertrophic stresses were significantly attenuated by Ad-SHP to cardiomyocytes. The expressions of hypertrophic markers such as ANF, B-type natriuretic peptide (BNP), skeletal 慣-actin and 慣-tubulin were significantly higher in hearts of SHP null mice than those in wild type mice hearts. SHP physically interacted with GATA6 in mammalian cells. SHP significantly decreased the activation of -3003 Nppa promoter induced by GATA6 in H9c2, cardiomyoblast cells. The action of SHP on Nppa promoter activity was partially recovered by GATA6. Conclusions: Taken together, these results suggest that SHP can play an important role as a novel anti-hypertrophic transcriptional regulator in the development of cardiac hypertrophy.