<title>Abstract</title><italic>Theileria equi</italic>, an intraerythrocytic protozoan parasite, causes equine piroplasmosis, a disease which negatively impacts the global horse industry. Genetic manipulation is one of the research tools under development as a control method for protozoan parasites, but this technique needs to be established for <italic>T</italic>. <italic>equi</italic>. Herein, we report on the first development of a stable transgenic <italic>T</italic>. <italic>equi</italic> line expressing enhanced green fluorescent protein/blasticidin S deaminase (eGFP/BSD). To express the exogenous fusion gene in <italic>T</italic>. <italic>equi</italic>, regulatory regions of the <italic>elongation factor-1 alpha</italic> (<italic>ef-1α</italic>) gene were identified in <italic>T. equi</italic>. An eGFP/BSD-expression cassette containing the <italic>ef-1α</italic> gene promoter and terminator regions was constructed and integrated into the <italic>T</italic>. <italic>equi</italic> genome. On day 9 post-transfection, blasticidin-resistant <italic>T</italic>. <italic>equi</italic> emerged. In the clonal line of <italic>T. equi</italic> obtained by limiting dilution, integration of the eGFP/BSD-expression cassette was confirmed in the designated B<italic>-</italic>locus of the <italic>ef-1α</italic> gene via PCR and Southern blot analyses<italic>.</italic> Parasitaemia dynamics between the transgenic and parental <italic>T</italic>. <italic>equi</italic> lines were comparable in vitro. The eGFP/BSD-expressing transgenic <italic>T</italic>. <italic>equi</italic> and the methodology used to generate it offer new opportunities for better understanding of <italic>T</italic>. <italic>equi</italic> biology, with the add-on possibility of discovering effective control methods against equine piroplasmosis.