The early branching eukaryote Entamoeba histolytica is a human parasite that is the etiologic agent of amebic dysentery and liver abscess. The sequencing of the E. histolytica genome combined with the development of an E. histolytica microarray has resulted in the identification of several distinct gene expression profiles associated with virulence. The function of many modulated transcripts is unknown and their role in pathogenicity is unclear. They however represent a pool of potential virulence factors that could be targets for the development of novel therapeutics. Efficient tools and methods to characterize these novel virulence-associated genes and proteins would be beneficial. Here we report the use of the Gateway((R)) cloning system to generate the E. histolytica expression vector pAH-DEST. To test the usefulness of this system, the vector was used to construct a plasmid containing a recombinant version of the locus EHI_144490, which encoded a protein of unknown function. The recombinant gene was expressed and the recombinant protein, which was strep-myc-tagged, showed a cytoplasmic localization in transfected trophozoites. This expression vector with the Gateway((R)) system should facilitate investigation into the functions of novel proteins in E. histolytica.