The subcellular localization of the human Ca2+‐binding EF‐hand/leucine zipper protein NEFA was studied in HeLa cells by immunofluorescence microscopy. Double immunostaining using mouse anti‐NEFA monoclonal antibody 1H8D12 and rabbit anti‐ERD2 polyclonal antibody proved that NEFA is localized in the Golgi apparatus. The result was confirmed by the expression of NEFA–green fluorescent protein (GFP) fusion protein in the Golgi in the same cell line. Cycloheximide treatment proved NEFA to be a Golgi‐resident protein. Seven NEFA deletion mutants were constructed to ascertain the peptide region relevant for Golgi retention. The expression of each NEFA–GFP variant was detected by fluorescence microscopy and immunoblotting. Only the ΔN mutant, lacking the N‐terminal Leu/Ile‐rich region, failed to be retained in the Golgi after cycloheximide treatment. The other six deletion mutants in which either the basic region, the complete EF‐hand pair domain, the two EF‐hand motifs separately, the leucine zipper and the leucine zipper plus the C‐terminal region is deleted, were localized to the Golgi. The peptide sequence within the Leu/Ile‐rich region is discussed as a novel Golgi retention motif.