The electrogenic sodium/calcium exchanger (NCX) mediates bidirectional calcium transport controlled by the transmembrane sodium gradient. NCX inactivation occurs in the absence of phosphatidylinositol 4,5-bisphosphate and is facilitated by palmitoylation of a single cysteine at position 739 within the large intracellular loop of NCX. The aim of this investigation was to identify the structural determinants of NCX1 palmitoylation. Full-length NCX1 (FL-NCX1) and a YFP fusion protein of the NCX1 large intracellular loop (YFP-NCX1) were expressed in HEK cells. Single amino acid changes around Cys-739 in FL-NCX1 and deletions on the N-terminal side of Cys-739 in YFP-NCX1 did not affect NCX1 palmitoylation, with the exception of the rare human polymorphism S738F, which enhanced FL-NCX1 palmitoylation, and D741A, which modestly reduced it. In contrast, deletion of a 21-amino acid segment enriched in aromatic amino acids on the C-terminal side of Cys-739 abolished YFP-NCX1 palmitoylation. We hypothesized that this segment forms an amphipathic α-helix whose properties facilitate Cys-739 palmitoylation. Introduction of negatively charged amino acids to the hydrophobic face or of helix-breaking prolines impaired palmitoylation of both YFP-NCX1 and FL-NCX1. Alanine mutations on the hydrophilic face of the helix significantly reduced FL-NCX1 palmitoylation. Of note, when the helix-containing segment was introduced adjacent to cysteines that are not normally palmitoylated, they became palmitoylation sites. In conclusion, we have identified an amphipathic α-helix in the NCX1 large intracellular loop that controls NCX1 palmitoylation. NCX1 palmitoylation is governed by a distal secondary structure element rather than by local primary sequence.