Calcineurin, a Ca2+-regulated protein phosphatase, links myocardial Ca2+ signaling with hypertrophic gene transcription. Calcineurin abundance increases in pressure-overload hypertrophy and may reduce agonist-mediated phospholamban (PLB) phosphorylation to underlie blunted β-adrenergic receptor (β-AR) responsiveness in hypertension. This hypothesis was tested by measuring the effects of calcineurin inhibition on changes in cardiac contractility caused by β-adrenergic stimulation in spontaneously hypertensive rats (SHR). Female SHR (age: 7 mo) and age-matched female Wistar-Kyoto rats (WKY) were studied. Heart weight-to-body weight ratio ( P < 0.01) and systolic blood pressure ( P < 0.01) were greater in SHR compared with WKY and were associated with increased myocardial calcineurin mRNA (CnAβ) and activity ( P < 0.05). β-AR stimulation with isoproterenol (Iso) increased calcineurin activity ( P < 0.05) in both WKY and SHR hearts, and this activity was suppressed with cyclosporin A (CsA) treatment. In SHR, CsA improved left ventricular whole heart and isolated myocyte β-AR responsiveness by normalizing PLB phosphorylation at Ser16 and Thr17 ( P < 0.05). These CsA-induced, PLB-mediated effects were associated with an augmentation in cardiomyocyte peak Ca2+ and a reduced rate (time constant of isovolumic pressure relaxation, tau) and magnitude of diastolic Ca2+ during β-AR stimulation. In conclusion, CsA normalized the blunted β-AR responsiveness associated with hypertension, in part, by mitigating calcineurin activity while improving PLB phosphorylation and subsequent sarcoplasmic reticulum Ca2+ regulation.