Muscle contractility is regulated by a network of many proteins. In cardiomyocytes, the sarco(endo)plasmic reticulum Ca2+ -ATPase, SERCA, and its regulatory protein, phospholamban are responsible for ∼70% of Ca2+ reuptake into the SR. While unphosphorylated, PLN inhibits SERCA by lowering its apparent Ca2+ affinity. Upon phosphorylation by PKA at Ser16, PLN inhibition is relieved. This tightly regulated interaction can be easily disrupted by mutation or changes in protein level, leading to heart disease. Thus, understanding the molecular interactions between SERCA/PLN and possible regulators is essential.Here, we report that ssDNA binds the cytoplasmic domain of PLN with low nanomolar dissociation constants, relieving inhibition of SERCA. The relief of inhibition is length dependent, while affinity is constant for oligonucleotides longer than 10 bases. Solution and solid-state NMR experiments have provided residue specific information that ssDNA targets the cytoplasmic domain of PLN and does not affect SERCA in the absence of PLN. In-cell FRET, and NMR experiments determined that addition of ssDNA does not dissociate PLN from SERCA.SERCA/PLN has become a highly targeted complex for development of small molecule regulators because of its prevalence in many cardiovascular diseases. While some therapies are currently being investigated, none have proceeded past clinical trials. These results provide a promising avenue for development of novel regulators of the SERCA/PLN complex. Additionally, they support previous findings from our group detailing the intricate balance that is necessary for proper cardiac function.