Sarcolipin (SLN) inhibits the cardiac sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA2a) by direct binding and is superinhibitory if it binds as a binary complex with phospholamban (PLN). To demonstrate whether overexpression of SLN in the heart might impair cardiac function directly, transgenic (TG) mice with cardiac-specific overexpression of NF-SLN (SLN tagged at its N terminus with the FLAG epitope) were generated on a phospholamban (PLN) null (PLN KO) background. In NF-SLN TG/PLN KO cardiac microsomes, the apparent affinity of SERCA2a for Ca2+ was decreased compared with non-TG littermate PLN KO hearts. Analyses of isolated NF-SLN/PLN KO cardiomyocytes revealed impaired cardiac contractility, reduced calcium transient peak amplitude, and slower decay kinetics compared to PLN KO animals. In these cardiomyocytes, isoproterenol restored calcium dynamics to the levels seen in PLN KO. Invasive hemodynamic and echocardiographic analyses of NF-SLN/PLN KO mouse cardiac muscle in vivo showed no direct effects of NF-SLN overexpression when compared to PLN KO mice. A possible mechanism for the lack of effects in the whole heart may be a responsiveness to phosphorylation because we determined that NF-SLN can be phosphorylated in cardiomyocytes in response to isoproterenol, and we provide evidence that serine/threonine kinase 16 is a kinase that can phosphorylate NF-SLN. Site-directed mutagenesis showed that SLN Thr-5 is the target site for this kinase. These data show that overexpression of NF-SLN can inhibit SERCA2a in the absence of PLN and that the inhibition of SERCA2a is correlated with impairment of contractility and calcium cycling in cardiomyocytes.