The trace metal zinc is essential in all types of organisms, where it has many catalytic, structural and regulatory functions. Zinc homeostasis in cells and organelles is maintained by various types of zinc transport protein. These include Cation Diffusion Facilitator (CDF) family proteins, which export zinc to the extracellular space or to the cytoplasm. Homologous CDF proteins are found in both prokaryotes and eukaryotes, where the human variants are the ZnTs or SLC30 family. One of the first and best characterised prokaryotic CDFs is the Escherichia coli zinc exporter ZitB, which is driven by the proton motive force in an antiport manner. In this article we provide an analytical review and expand on the biochemical and computational characterisation of ZitB and assess its potential for high-resolution three-dimensional structure determination. Consistent with structures determined for other CDF proteins (YiiP, ZnTs 3, 4, 7 and 8), the 313 residues of ZitB are predicted to form six transmembrane spanning α-helices with a long cytoplasmic C-terminal tail. An unusual feature of ZitB is an exceptionally high (8.0%) content of histidine residues. Using the IPTG-inducible plasmid pTTQ18, we demonstrate the cloning and amplified expression in E. coli of non-tagged, wild-type ZitB at levels of ~15% of total protein in preparations of inner membranes. ZitB was solubilised in the mild detergent n-dodecyl-β-D-maltoside (DDM) and purified by immobilised metal affinity chromatography in yields of ~1.8 mg per litre of culture medium. The structural integrity of purified ZitB was confirmed by mass spectrometry and circular dichroism spectroscopy.