17-Methoxyl-7-hydroxy-benzene-furanchalcone (MHBFC) was isolated from the Millettia pulchra (Benth.) Kurz var. Laxior (Dunn) Z. Wei (Papilionaceae) (MKL) roots. This study aimed to study the effects of MHBFC on cardiac hypertrophy after banding of the abdominal aorta in wild-type mice and endothelial nitric oxide synthase (eNOS) knockout mice in order to verify whether MHBFC depends on the eNOS gene in reversing cardiac hypertrophy. By observing the blood pressure, body weight, ventricular hypertrophy index, expression of B-type natriuretic peptide (BNP), myosin heavy chain beta (β-MHC), atrial natriuretic peptide (ANP), serum amyloid A SAA mRNA, level of endothelin-1 (ET-1) protein, histopathological changes of myocardial cells, and the myocardial ultrastructure, we found that MHBFC significantly ameliorated the changes in the structure of the heart and aorta, significantly decreased the blood pressure, myocardial cell cross-section area, level of ET-1 protein, and mRNA levels of BNP, β-MHC, ANP and SAA, prevented myocardial ultrastructure alterations, and relieved myocardial and perivascular fibrosis of wild-type mice. However, MHBFC could not induce any significant change in eNOS gene knockout mice. These results show that MHBFC is beneficial to protect against cardiac hypertrophy after banding of the abdominal aorta in wild-type mice, but has no such effect on eNOS gene knockout mice, which indicates that MHBFC is dependent on the existence of the eNOS gene in reversing cardiac hypertrophy.