Despite the huge efforts and the medical interest in heart failure, it remains a common deadly disease with an associated high health cost in the western world. Alteration in calcium homeostasis during excitation–contraction coupling has been recognised in many studies associated with heart failure. In the heart the plasma membrane calcium ATPase (PMCA) had originally been thought to play a rather minor role in cardiac relaxation. However, our group has suggested a new function for isoform 4 of PMCA in the heart as a modulator of signal transduction pathways through its interaction with neuronal nitric oxide synthase (nNOS). Importantly, the PMCA4/nNOS complex has been found to ablate the β-adrenergic inotropic response in the heart (Oceandy et al, 2007). In the current study, we aim to investigate the mechanisms by which PMCA4 regulates β-adrenergic response through nNOS modulation. We used whole hearts and adult cardiomyocytes isolated from transgenic mice overexpressing PMCA4 in the heart, as well as neonatal rat cardiomyocytes (NRCM) with adenoviral overexpression of PMCA4. PMCA4 transgenic animals showed a 75% reduction in nNOS activity and a 50% reduction in total nitric oxide synthase activity compared with their wild-type (WT) littermates (n = 8, p<0.05), as well as a 25% reduction in cyclic guanosine monophosphate (cGMP) levels compared with WT littermates (n = 8, p<0.05). In contrast, PMCA4 transgenic hearts showed 30% elevation in cyclic adenosine monophosphate (cAMP) levels compared with their WT littermates (n = 8, p<0.05). Further analysis using adult cardiomyocytes isolated from PMCA4 transgenic mice demonstrated that the PMCA4 transgenic myocytes showed threefold higher Ser16-phospholamban (PLB) as well as Ser22&23-cardiac troponin-I (cTnI) phosphorylation at baseline compared with WT myocytes. In addition, the relative induction of phospholamban and cTnI phosphorylation following isoprenaline treatment was severely reduced in PMCA4 transgenic explaining the blunted physiological response to β-adrenergic stimulation. In keeping with the data from transgenic animals, NRCM overexpressing PMCA4 showed a significant reduction by 21.4 ± 5.11% in nitric oxide level and 24 ± 5.09% in cGMP levels compared with control cells (n = 6 each and p<0.05). In addition, PMCA4 overexpression increased cAMP levels by 94.4 ± 8.17% compared with controls (n = 6, p<0.05). Overexpression of PMCA4 in NRCM increased Ser16-PLB as well as Ser22&23-cTnI by twofold compared with the control at baseline. We also found that the regulation of cardiac phosphodiesterase activity determined the balance between cGMP and cAMP following PMCA4 overexpression. These results showed that the nNOS/PMCA4 complex regulates contractility via cAMP and phosphorylation of phospholamban and troponin-I. Overall, these findings provide mechanistic insight into the regulation of the β-adrenergic response in the heart by PMCA4 and place this calcium pump upstream of the pathway linking nNOS signalling and Ser16-PLB phosphorylation and downstream of the β-adrenergic receptor(s).