Phosphorylation at serine 68 of phospholemman (PLM) in response to β-adrenergic stimulation results in simultaneous inhibition of cardiac Na+/Ca2+exchanger NCX1 and relief of inhibition of Na+-K+-ATPase. The role of PLM in mediating β-adrenergic effects on in vivo cardiac function was investigated with congenic PLM-knockout (KO) mice. Echocardiography showed similar ejection fraction between wild-type (WT) and PLM-KO hearts. Cardiac catheterization demonstrated higher baseline contractility (+dP/d t) but similar relaxation (−dP/d t) in PLM-KO mice. In response to isoproterenol (Iso), maximal +dP/d t was similar but maximal −dP/d t was reduced in PLM-KO mice. Dose-response curves to Iso (0.5–25 ng) for WT and PLM-KO hearts were superimposable. Maximal +dP/d t was reached 1–2 min after Iso addition and declined with time in WT but not PLM-KO hearts. In isolated myocytes paced at 2 Hz. contraction and intracellular Ca2+concentration ([Ca2+]i) transient amplitudes and [Na+]ireached maximum 2–4 min after Iso addition, followed by decline in WT but not PLM-KO myocytes. Reducing pacing frequency to 0.5 Hz resulted in much smaller increases in [Na+]iand no decline in contraction and [Ca2+]itransient amplitudes with time in Iso-stimulated WT and PLM-KO myocytes. Although baseline Na+-K+-ATPase current was 41% higher in PLM-KO myocytes because of increased α1- but not α2-subunit activity, resting [Na+]iwas similar between quiescent WT and PLM-KO myocytes. Iso increased α1-subunit current ( Iα1) by 73% in WT but had no effect in PLM-KO myocytes. Iso did not affect α2-subunit current ( Iα2) in WT and PLM-KO myocytes. In both WT and NCX1-KO hearts, PLM coimmunoprecipitated with Na+-K+-ATPase α1- and α2-subunits, indicating that association of PLM with Na+-K+-ATPase did not require NCX1. We conclude that under stressful conditions in which [Na+]iwas high, β-adrenergic agonist-mediated phosphorylation of PLM resulted in time-dependent reduction in inotropy due to relief of inhibition of Na+-K+-ATPase.