The anti-apoptotic protein HAX-1 has been proposed to modulate cardiac calcium handling by interacting with the sarcoendoplasmic reticulum calcium ATPase (SERCA) and its regulatory partner, phospholamban (PLB). HAX-1 is thought to enhance the inhibitory regulation of SERCA by phospholamban, but the mechanism of this functional effect is unknown. Here we used fluorescence resonance energy transfer (FRET) microscopy to quantify the effects of HAX-1 on the structure and affinity of the SERCA-PLB regulatory complex. In parallel experiments, HAX-1 was either coexpressed with Cer-SERCA and YFP-PLB in HEK cells, or recombinant HAX-1 was applied to saponin-permeabilized cells expressing Cer-SERCA and YFP-PLB. We observed that coexpression of HAX-1 significantly increased SERCA and PLB protein expression in HEK cells, consistent with its role as an anti-apoptotic factor. Despite this, we observed a modest decrease in FRET from SERCA to PLB, consistent with a small change in the quaternary structure of the regulatory complex. This effect was dose-dependent, with increasing expression of HAX-1 further decreasing intrinsic FRET efficiency from 31% toward a minimum of 26%. We did not observe a change in the binding affinity of PLB for SERCA, since HAX-1 did not significantly change the apparent dissociation constant (KD) of the regulatory complex. Overall, the data are consistent with previous NMR studies that suggested HAX-1 binds to the PLB cytoplasmic domain and increases its association with the surface of the bilayer membrane.