Activation of NF-κB contributes to cardiac hypertrophy and the interleukin-1 receptor (IL-1R)-mediated MyD88-dependent signaling pathway predominately activates NF-κB. Recent studies have shown that the TIR/ BB-Loop mimetic (AS-1) disrupted the interaction of MyD88 with the IL-1R, resulting in blunting of NF-κB activation. We have examined the effects of AS-1 on the IL-1b-induced hypertrophic response using cultured neonatal cardiac myocytes in vitro and transverse aortic constriction (TAC) pressure overload-induced cardiac hypertrophy in vivo. Neonatal cardiac myocytes were treated with AS-1 15 min prior to IL-1β stimulation for 24 h. AS-1 treatment significantly attenuated IL-1β-induced hypertrophic responses of cardiac myocytes. In vivo experiments showed that AS-1 administration prevented cardiac hypertrophy and dysfunction induced by pressure overload. AS-1 administration disrupted the interaction of IL-1R with MyD88 in the pressure overloaded hearts and prevented activation of NF-κB. In addition, AS-1 prevented increases in activation of the MAPK pathway (p38 and p-ERK) in TAC-induced hypertrophic hearts. Our data suggest that the IL-1R-mediated MyD88-dependent signaling pathway plays a role in the development of cardiac hypertrophy and AS-1 attenuation of cardiac hypertrophy is mediated by blocking the interaction between IL-1R and MyD88, resulting in decreased NF-κB binding activity and decreased MAPK activation.