Sarcoplasmic/endoplasmic reticulum Ca2+-ATPase1 (SERCA1) is responsible for the clearance of cytosolic Ca2+ in skeletal muscle, and its regulation has been intensively studied. Small ankyrin 1 (sAnk1, Ank1.5), a 17 kDa muscle-specific isoform of ANK1, binds to SERCA1 directly via both its transmembrane and cytoplasmic domains and inhibits SERCA1’s ATPase activity. It also binds avidly to the giant cytoskeletal protein, obscurin. We characterized the interaction between the cytoplasmic domain of sAnk1 (sAnk1(29-155)) and SERCA1. The binding affinity was 444 nM by blot overlay. Site-directed mutagenesis identified K38, H39, and H41, in the juxtamembrane region, as essential for binding to SERCA1 but not to obscurin. Residues R64-K73, which contribute to obscurin binding, are also required for binding to SERCA1, but only the hydrophobic residues in this sequence are required, not the positively charged residues necessary for obscurin binding. Circular dichroism analysis of sAnk1(29-155) indicates that most mutants we studied show significant structural changes, with the exception of K38A/H39A/H41A. The inhibitory activity of full-length sAnk1 requires the WT juxtamembrane sequence. Our results suggest that, in addition to its transmembrane domain, sAnk1 uses its juxtamembrane sequence and perhaps part of its obscurin binding site to bind to SERCA1, and that this binding contributes to their robust association in situ, as well as regulation of SERCA1’s activity. In addition, we showed sAnk1 binds directly to PLN and forms a three-way complex with SERCA1. This interaction does not ablate PLN’s inhibition of SERCA1 activity. Our results are consistent with a model in which, in forming a three-way complex, PLN binds to SERCA1 first, followed by binding to sAnk1. Our studies with PLN suggest that the interactions of PLN, SLN, and sAnk1 with SERCA1, either alone or in pairs, are distinct and have different effects on SERCA1’s enzymatic activity