Mitogen-activated protein kinase kinase 7 (MKK7) induces cardiac hypertrophy by activating the c-Juns NH2-terminal kinases (JNK). It has been reported that growth arrest and DNA-damage-inducible beta (GADD45Β) binds to MKK7 directly and blocks its catalytic activity, mediates the inhibition of JNK signaling. However, the potential role of GADD45Β on cardiac hypertrophy has not been investigated. In this study, we found co-infection of cardiomyocytes with adenoviral vectors expressing MKK7 and GADD45B could counteract the characteristic hypertropic responses, including an increase in cell size and elevated atrial natriuretic factor (ANP) expression which induced by overexpression of MKK7. Furthermore, siRNA-mediated knockdown of GADD45B could also cause cardiomyocytes hypertrophy. GeneChip data showed that GADD45B mRNA decreased significantly in patients with hypertrophy cardiomyopathy (HCM) compared with healthy subjects. Association study indicated that haplotype (rs2024144–rs3783501) of GADD45B affected the thickness of inter-ventricular septum in patients with HCM. Dual-luciferase assay showed that C–A haplotype displayed significantly increased transcription activity compared to T–G haplotype.