We investigated the role of inducible NOS (iNOS) on cardiac function during the development of left ventricular hypertrophy. Hypertrophy was induced by pressure-overload via short-term (2.5 months) or long-term (6.5 months) aortic banding (AoB) in wild-type (WT) and iNOS knock out (iNOSKO) mice. Cardiac function was then assessed via echocardiography, in situ hemodynamics and papillary muscle force measurements. Quantitative RT-PCR and Western blots were used to measure expression of hypertrophic gene markers and proteins respectively. Our data demonstrate that increased afterload via AoB leads to increased expression of iNOS that is associated with cardiac dysfunction. In pressure-overload induced hypertrophy, iNOSKO delays both the expression of hypertrophic markers and contractile dysfunction without causing significant changes in the level of hypertrophy. Moreover, after long-term AoB, iNOSKO animals exhibited increased basal cardiac function and an improved response to beta-adrenergic stimulation compared to long-term AoB WT animals. In conclusion, our data demonstrate that NO production via iNOS plays an important role in modulating cardiac function after moderate AoB that mimics long-term hypertension in humans.