Objective: The aim of this study was to select the best medium to maintain sperm motility during sperm-DNA incubation and assess the DNA uptake by spermatozoa of Iranian Holstein bulls and its effects on sperm motility. Materials and Methods: frozen sperms from an Iranian Holstein bull were thawed and centrifuged. Motile sperms were separated through Puresperm gradient (40/80%) followed by two times washing in SP-TALP medium. Then, sperms were washed once (PBS, Opti-MEM and SP-TALP) and incubated with DNA in each media followed by sperm motility estimation. The plasmid pEGFP-C1 was linearized and incubated with sperms at 37°C for 1 hour. Sperm-DNA mixture was treated with DNase I and the sperm pellet was washed with PBS. DNA extraction from sperms and supernatants from the last washing were used as template for PCR. Data was analyzed using SAS package and mean comparisons between sperm motility in different media were performed. Results: Sperm motility after incubation in PBS, Opti-MEM and SP-TALP were 40(±2.89), 2(±1.53) and 54(±4.41) percent, respectively. PCR results from transfected sperms indicated that EGFP transgene internalized into the bovine sperms and DNaseI treatment could not eliminate it. Conclusion: In conclusion the best medium for sperm and DNA incubation was SPTALP. The DNA not only could attach to the post acrosomal region of spermatozoa but also could integrate into it. So bovine spermatozoa can be used as transgene carrier into oocyte.