Phospholemman (FXYD1, or PLM) is a key regulator of Na+-K+ ATPase in the heart, and is principally phosphorylated by cAMP-dependent protein kinase A (PKA) and protein kinase C (PKC). To investigate whether phosphorylation alters FXYD1 structure and oligomerization, we fused cyan or yellow fluorescent protein (CFP/YFP) to the c-terminus of FXYD1 and co-expressed the fusion proteins in AAV-293 cells. Phosphorylation of FXYD1 was mimicked by mutations S68E (PKA site) or S63E/S68E (PKC+PKA sites), and FRET from CFP-FXYD1 to YFP-FXYD1 was quantified by acceptor photobleaching. FRET increased with protein concentration up to a maximum (FRETmax), which was taken to represent the intrinsic FRET of the bound complex. We did not detect significant changes of FRETmax with phosphomimetic mutations, suggesting the quaternary structure of FXYD1 oligomer is not grossly altered by phosphorylation. The concentration dependence of FRET also yielded the relative dissociation constant of the FXYD1 oligomer (Kd), in arbitrary units (AU). Compared to non-phosphorylatable mutant S68A, S68E showed a significant decrease in Kd (14.1 ± 2.0 and 7.3 ± 2.0 AU, respectively). The data are consistent with more avid oligomerization of pseudo-phosphorylated FXYD1. Phosphomimetic mutation of both PKC and PKA sites (S63E/S68E) resulted in a Kd of 6.6 ± 1.1 AU, suggesting there was not a significant additional increase in oligomerization vs. the single site mutation. Taken together, the data suggest that phosphorylation can enhance FXYD1 oligomerization without altering the architecture of the oligomeric complex. Increased FXYD1 oligomerization may have an indirect effect on the regulatory interaction of FXYD1 with Na+-K+ ATPase.