Osteopontin (OPN) is upregulated in several experimental models of cardiac fibrosis and remodeling. However, its direct effects remain unclear. We examined the hypothesis that OPN is important for the development of cardiac fibrosis and remodeling. Moreover, we examined whether the inhibitory effect of eplerenone (Ep), a novel aldosterone receptor antagonist, was mediated through the inhibition of OPN expression against cardiac fibrosis and remodeling. Wild-type (WT) and OPN-deficient mice were treated with angiotensin II (Ang II) for 4 weeks. WT mice receiving Ang II were divided into 2 groups: a control group and an Ep treatment group. Ang II treatment significantly elevated blood pressure and caused cardiac hypertrophy and fibrosis in WT mice. Ep treatment and OPN deficiency could reduce the Ang II–induced elevation of blood pressure and ameliorate the development of cardiac fibrosis, whereas Ep-only treatment abolished the development of cardiac hypertrophy. Most compelling, the reduction of cardiac fibrosis led to an impairment of cardiac systolic function and subsequent left ventricular dilatation in Ang II–treated OPN-deficient mice. These results suggest that OPN has a pivotal role in the development of Ang II–induced cardiac fibrosis and remodeling. Moreover, the effect of Ep on the prevention of cardiac fibrosis, but not cardiac hypertrophy, might be partially mediated through the inhibition of OPN expression.