Phospholemman (PLM) regulates cardiac Na⁺/Ca²⁺ exchanger (NCX1) and Na⁺-K⁺-ATPase in cardiac myocytes. PLM, when phosphorylated at Ser⁶⁸, disinhibits Na⁺-K⁺-ATPase but inhibits NCX1. PLM regulates cardiac contractility by modulating Na⁺-K⁺-ATPase and/or NCX1. In this study, we first demonstrated that adult mouse cardiac myocytes cultured for 48 h had normal surface membrane areas, t-tubules, and NCX1 and sarco(endo)plasmic reticulum Ca²⁺-ATPase levels, and retained near normal contractility, but α₁-subunit of Na⁺-K⁺-ATPase was slightly decreased. Differences in contractility between myocytes isolated from wild-type (WT) and PLM knockout (KO) hearts were preserved after 48 h of culture. Infection with adenovirus expressing green fluorescent protein (GFP) did not affect contractility at 48 h. When WT PLM was overexpressed in PLM KO myocytes, contractility and cytosolic Ca²⁺ concentration ([Ca²⁺]_i) transients reverted back to those observed in cultured WT myocytes. Both Na⁺-K⁺-ATPase current (I_pump) and Na⁺/Ca²⁺ exchange current (I_NaCa) in PLM KO myocytes rescued with WT PLM were depressed compared with PLM KO myocytes. Overexpressing the PLMS68E mutant (phosphomimetic) in PLM KO myocytes resulted in the suppression of I_NaCa but had no effect on I_pump. Contractility, [Ca²⁺]_i transient amplitudes, and sarcoplasmic reticulum Ca²⁺ contents in PLM KO myocytes overexpressing the PLMS68E mutant were depressed compared with PLM KO myocytes overexpressing GFP. Overexpressing the PLMS68A mutant (mimicking unphosphorylated PLM) in PLM KO myocytes had no effect on I_NaCa but decreased I_pump. Contractility, [Ca²⁺]_i transient amplitudes, and sarcoplasmic reticulum Ca²⁺ contents in PLM KO myocytes overexpressing the S68A mutant were similar to PLM KO myocytes overexpressing GFP. We conclude that at the single-myocyte level, PLM affects cardiac contractility and [Ca²⁺]_i homeostasis primarily by its direct inhibitory effects on Na⁺/Ca²⁺ exchange.