Cardiac hypertrophy is characterized by increased cardiomyocyte size including physiological and pathological hypertrophy. Doxorubicin (DOX) widely used as the most potent anthracycline antibiotic agents for a wide range of cancers. DOX is induce cardiac hypertrophy because of it has a dose-limiting side effect such as dilated cardiomyopathy and lipid peroxidation. TongGuanWan (TGW, 通關丸), a well-known traditional herbal formula, has been reported that delay atherosclerotic formation and development and improving diabetes. which is most closely related to diabetes mellitus. However, the protective effect of TGW on cardiac hypertrophy and fibrosis was not investigated. The aim of this study was to investigate the effects of TGW on cardiac hypertrophy and fibrosis, which has been implicated in heart failure in H9c2 cells and isoproterenol (ISO)-induced cardiac hypertrophy mice. In vitro, H9c2 cells were induced by DOX (1 μM) in the presence or absence of TGW (1–10 μg/ml) and incubated for 24 h. In vivo, TGW was administrated to ISO-induced cardiac hypertrophy mice (n=8) to 100 and 200 mg/kg/day concentrations, respectively. The experiment was measured for 2 weeks following TGW respectively. The results of in vitro study indicated that TGW inhibit the increase cell surface area and the upregulation of hypertrophy markers including atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), beta-myosin heavy chain (β-MHC), and Myosin Light Chain-2 (MLC2) in H9c2 cells. In addition, TGW reduced DOX-induced the MAPKs protein expression and NF-κB p65 activation in H9c2 cells. TGW treatment remarkably inhibit activation of GATA-4/calcineurin/NFAT-3 signaling pathway under DOX conditions. Moreover, TGW significantly reduce DOX-induced expression of cell apoptosis-related proteins and fibrosis biomarkers. The results of in vivo study indicated that TGW significantly reduced the heart weight mass to body weight ratio (HW/BW) and left ventricle mass to body weight ratio (LV/BW) levels compared to the untreated the ISO-induced cardiac hypertrophy mice. Administration of TGW reduced protein expression of hypertrophy markers in cardiac hypertrophy mice. Moreover, TGW showed decreased that perivascular fibrosis and vessel hypertrophy in the heart tissues of cardiac hypertrophy mice, as determined by Picro-Sirius Red staining. TGW group significantly inhibited protein and mRNA expression levels of fibrosis related factors such as fibronectin, α-SMA and collagen type I compared to the untreated the ISO-induced cardiac hypertrophy group. Therefore, these results suggest that TGW may be potential therapies targeting the prevention and cure of cardiac hypertrophy and fibrosis, which finally causes sudden heart failure.