Sarcoplasmic/endoplasmic reticulum Ca2+-ATPase1 (SERCA1) is responsible for the clearance of cytosolic Ca2+ in skeletal muscle. Due to its vital importance in regulating Ca2+ homeostasis, the regulation of SERCA1 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. In this study, we first characterize the interaction between the cytoplasmic domain of sAnk1 (sAnk1(29-155)) and SERCA1. The binding affinity for sAnk1 (29-155) to SERCA1 is 444 nM by blot overlay, about 7-fold weaker than the binding of sAnk1(29-155) to obscurin, a giant protein of the muscle cytoskeleton. Site-directed mutagenesis identifies K38, H39, and H41, in the juxtamembrane region, as unique binding sites for SERCA1. 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 show significant structural changes, with the exception of those containing alanines in place of K38, H39 and H41. Although the cytoplasmic domain of sAnk1 does not inhibit SERCA1’s Ca2+-ATPase activity, with or without mutations in the juxtamembrane sequence, the inhibitory activity of full-length sAnk1 requires the wild-type (WT) juxtamembrane sequence. We used these data to model sAnk1 and the sAnk1-SERCA1 complex. 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. We have previously shown that sAnk1 binds to sarcolipin (SLN), a 31-amino acid peptide inhibitor of SERCA1, forming a three-way complex with SERCA1. Notably, the presence of sAnk1 ablates SLN’s inhibitory effect on SERCA1. Based on this, we examined the binding of sAnk1 to another well-known SERCA inhibitor, phospholamban (PLN). Here we use co-transfection of COS7 cells, co-immunoprecipitation (Co-IP), and bimolecular fluorescent complementation (BiFC) to show that sAnk1 associates with PLN and forms a 3-way complex with PLN and SERCA1. Anisotropy-based FRET (AFRET) studies of Cerulean-SERCA1 with Venus-tagged sAnk1 and PLN, as well as with the Venus tag formed by these two proteins through BiFC, confirms the presence of a 3-way complex. ATPase assays show that, unlike its effects on SLN, sAnk1 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. This modeling suggests 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. Finally, we report a direct interaction between the C-terminus of obscurin and SERCA1, with an affinity of 1.09 μM in blot overlay assays. Our data suggests that, sAnk1(R64-K73) may dock to obscurin and SERCA1 simultaneously via different residues, potentially forming a novel three-way complex. This interaction raises the possibility that SERCA can be dynamically regulated during muscle contraction. Further studies are required to confirm this.