The zinc/iron permease (ZIP/SLC39A) family plays an important role in metal ion transport and is essential for diverse physiological processes. Members of the ZIP family function primarily in the influx of transition metal ions zinc and iron, into cytoplasm from extracellular space or intracellular organelles. The molecular determinants defining metal ion selectivity among ZIP family members remain unclear. Specifically, we reported before that the Drosophila ZIP family member ZIP13 (dZIP13), functions as an iron exporter and was responsible for pumping iron into the secretory pathway. ZIP13 protein is unique in that it differs from the other LIV-1 subfamily members at transmembrane domain IV (TM4), wherein relative positions of the conserved H and D residues in the HNXXD sequence motif are switched, generating a DNXXH motif. In this study, we undertook an in vivo approach to explore the significance of this D/H exchange. Comparative functional analysis of mutants revealed that the relative positions of D and H are critical for the physiological roles of dZIP13 and its close homologue dZIP7. Swapping D/H position of this DNXXH sequence in dZIP13 resulted in loss of iron activity; normal dZIP13 could not complement dZIP7 loss, but swapping the two relative amino acid positions D and H in dZIP13 was sufficient to make it functionally analogous to its close homologue dZIP7. This work provides the first in vivo functional analysis of a structural motif required to differentiate different transporting functions of ZIPs.