Phospholemman (FXYD1), abundantly expressed in the heart, is the primary cardiac sarcolemmal substrate for PKA and PKC. Evidence supports the hypothesis that phospholemman (PLM) is part of the cardiac Na/K pump complex and provides the link between kinase activity and pump modulation. PLM has also been proposed to modulate Na/Ca exchanger activity and may be involved in cell volume regulation. This study characterised the phenotype of the PLM knockout (KO) mouse heart, to further our understanding of PLM function in the heart. PLM KO mice were bred on a congenic C57/Bl6 background. In vivo conductance catheter measurements exhibited a mildly depressed cardiac contractile function in PLM KO mice, which was exacerbated when hearts were isolated and Langendorff-perfused. There were no significant differences in action potential morphology in paced Langendorff-perfused hearts. Depressed contractile function was associated with a mild cardiac hypertrophy in PLM KO mice. Biochemical analysis of crude ventricular homogenates showed a significant increase in Na/K ATPase activity in PLM KO hearts compared with wild-type controls. SDS-PAGE/Western blot analysis of ventricular homogenates revealed small, non-significant changes in Na/K ATPase subunit expression, with 2D-gel (IEF, SDS-PAGE) analysis revealing minimal changes in ventricular protein expression, indicating that deletion of PLM was the primary reason for the observed PLM KO phenotype. These studies demonstrate PLM plays an important role in the contractile function of the normoxic mouse heart. Data is consistent with the hypothesis that PLM modulates Na/K ATPase activity, indirectly affecting intracellular Ca and hence contractile function.