Transfection has been a valuable technique for elucidating gene function in many pathogens. While transient transfection of Babesia spp. has been reported previously, stable integration of exogenous genes in Babesia has proven difficult. In this study, a plasmid was designed to target integration of a gfp-bsd gene into the Babesia bovis ef-1alpha locus. Babesia bovis-infected erythrocytes of the biologically cloned Mo7 strain were transfected by electroporation with either circular or linear plasmids and selected in cultures with varying amounts of blasticidin 24h after electroporation. Several blasticidin-resistant B. bovis transfected cell lines emerged at different rates, ranging from 5 to 26 days after the start of selection. One transfected parasite line (1-2-124) was selected for further analysis based on a rapid growth rate and bright GFP fluorescence in the presence of a lethal concentration of blasticidin. Continued expression of the gfp-bsd fusion gene was confirmed by reverse transcriptase-PCR, Western blot analysis and fluorescence microscopy for longer than 9 months after electroporation. No plasmid or episomal DNA could be detected in this line, and plasmid recovery in Escherichia coli was unsuccessful. Southern blot results and sequencing of PCR amplicons flanking the putative insertion site are consistent with integration of at least one gfp-bsd cassette into the targeted ef-1alpha locus in the transfected parasite line. Overall the results demonstrate, we believe for the first time, chromosomal integration and stable expression of a foreign gene in B. bovis. With the availability of the B. bovis genome, targeted stable transfection will provide a means to determine the role of specific genes in the biology, clinical disease and immunity of B. bovis, one of the three major tick-borne parasites that limit global livestock production.