In this study, we evaluated whether blocking MyD88 could decrease cardiac myocyte apoptosis following pressure overload. Adenovirus expressing dominant negative MyD88 (Ad5-dnMyD88) or Ad5-GFP was transfected into rat hearts (n=8/group) immediately followed by aortic banding for 3 weeks. One group of rats (n=8) was subjected to aortic banding for three weeks without transfection. Sham surgical operation (n=8) served as control. The ratios of heart weight/body weight (HW/BW) and heart weight /tibia length (HW/TL) were calculated. Cardiomyocyte size was examined by FITC labeled wheat germ agglutinin staining of membranes. Cardiac myocyte apoptosis was determined by TUNEL assay and myocardial interstitial fibrosis was examined by Masson's Trichrome staining. Aortic banding significantly increased the ratios of HW/BW by 41.0% (0.44 ± 0.013 vs 0.31 ± 0.008), HW/TL by 47.2% (42.7 ± 1.30 vs 29.0 ± 0.69), and cardiac myocyte size by 49.6%, cardiac myocyte apoptosis by 11.5% and myocardial fibrosis, and decreased cardiac function compared with sham controls. Transfection of Ad5-dnMyD88 significantly reduced the ratios of HW/BW by 18.2% (0.36 ± 0.006 vs 0.44 ± 0.013), HW/TL by 22.3 % (33.2 ± 0.95 vs 42.7 ± 1.30), and decreased cardiomyocyte size by 56.8%, cardiac myocyte apoptosis by 76.2%, as well as fibrosis, and improved cardiac function compared with aortic banded group. Our results suggest that MyD88 is an important component in the TLR4 mediated NFB activation pathway that contributes to the development of cardiac hypertrophy. Blockade of MyD88 significantly reduced cardiac hypertrophy, cardiac myocyte apoptosis and improved cardiac function in vivo.