The guanine nucleotide binding protein Rab8A controls the final steps of exocytosis in mammalian cells. It has been implicated in the regulation of apical protein localization in intestinal epithelial cells and ciliary biogenesis. The in vitro structural and biochemical characterization of Rab8A and its interaction with regulator and effector molecules has been hampered by its insolubility in Escherichia coli expression systems. The conventional refolding procedure is laborious and yields only minute amounts of C-terminally truncated Rab8A (Rab8A1-183: amino acids 1183), not the full-length protein. Here, we report a method of expressing soluble, hexahistidine-tagged full-length human Rab8A from E. coli. The Rab8A gene was codon-optimized and coexpressed with bacterial GroEL and GroES chaperones. After two-step purification by Ni2+ affinity chromatography and gel filtration, Rab8A was obtained at a yield of 4 mg protein per 1 L of bacterial cell culture and a purity of >95%. The resultant protein was functionally active, as determined by GTPase activity and its interaction with the nucleotide exchange factor MSS4.