Phospholemman (PLM) belongs to the FXYD family of small ion transport regulators. When phosphorylated at serine68, PLM inhibits cardiac Na⁺/Ca²⁺ exchanger (NCX1). We previously demonstrated that the cytoplasmic tail of PLM interacts with the proximal intracellular loop (residues 218-358), but not the transmembrane (residues 1-217 and 765-938) or calcium binding (residues 371-508) domains of NCX1. In this study, we used intact Na⁺/Ca²⁺ exchanger with various deletions in the intracellular loop to map out the interaction sites with PLM. We first demonstrated by Western blotting and confocal immunofluorescence microscopy that wild-type (WT) NCX1 and its deletion mutants were expressed in transfected HEK293 cells. Co-transfection with PLM and NCX1 (or its deletion mutants) in HEK293 cells did not decrease NCX1 (or its deletion mutants) expression. Co-expression of PLM with wild-type NCX1 inhibited NCX1 current (I_NaCa). Deletion of residues 240-679, 265-373, 250-300 or 300-373 from WT NCX1 resulted in loss of inhibition of I_NaCa by PLM. Inhibition of I_NaCa by PLM was preserved when residues 229-237, 270-300, 328-330 or 330-373 were deleted from the intracellular loop of NCX1. These results suggest that PLM mediated inhibition of I_NaCa by interacting with two distinct regions (residues 238-270 and 300-328) of NCX1. Indeed, I_NaCa measured in mutants lacking residues 238-270, 300-328, or 238-270 + 300-328 was not affected by PLM. Glutathione S-transferase (GST) pull-down assays confirmed that PLM bound to fragments corresponding to residues 218-764, 218-371, 218-320, 218-270, 238-371 and 300-373, but not to fragments encompassing residues 250-300 and 371-508 of NCX1, indicating residues 218-270 and 300-373 physically associated with PLM. Finally, acute regulation of I_NaCa by PLM phosphorylation observed with WT NCX1 was absent in 250-300 but preserved in 229-237 deletion mutants. We conclude that PLM mediates its inhibition of NCX1 by interacting with residues 238-270 and 300-328.