Cardiac sympathetic stimulation activates β-adrenergic (β-AR) receptors and protein kinase A (PKA) phosphorylation of proteins involved in myocyte Ca regulation. The Na/K-ATPase (NKA) is essential in regulating intracellular [Na] ([Na]_i), which in turn affects [Ca]_i via Na/Ca exchange. However, how PKA modifies NKA function is unknown. Phospholemman (PLM), a member of the FXYD family of proteins that interact with NKA in various tissues, is a major PKA substrate in heart. Here we tested the hypothesis that PLM phosphorylation is responsible for the PKA effects on cardiac NKA function using wild-type (WT) and PLM knockout (PLM-KO) mice. We measured NKA-mediated [Na]_i decline and current (I_Pump) to assess β-AR effects on NKA function in isolated myocytes. In WT myocytes, 1 μmol/L isoproterenol (ISO) increased PLM phosphorylation and stimulated NKA activity mainly by increasing its affinity for internal Na (K_m decreased from 18.8±1.4 to 13.6±1.5 mmol/L), with no significant effect on the maximum pump rate. This led to a significant decrease in resting [Na]_i (from 12.5±1.8 to 10.5±1.4 mmol/L). In PLM-KO mice under control conditions K_m (14.2±1.5 mmol/L) was lower than in WT, but comparable to that for WT in the presence of ISO. Furthermore, ISO had no significant effect on NKA function in PLM-KO mice. ATPase activity in sarcolemmal vesicles also showed a lower K_m(Na) in PLM-KO versus WT (12.9±0.9 versus 16.2±1.5). Thus, PLM inhibits NKA activity by decreasing its [Na]_i affinity, and this inhibitory effect is relieved by PKA activation. We conclude that PLM modulates the NKA function in a manner similar to the way phospholamban affects the related SR Ca-ATPase (inhibition of transport substrate affinity, that is relieved by phosphorylation).