Background: Many studies conducted on the pentoxifylline (PX) and Glycyrrhiza glabra (G.glabra) as motility stimulants showed positive effect on sperm preparation in vitro improving the progressive forward movement. However, it was not known the impact of these stimulants on the nature of genetic material, especially after cryopreservation. Objective: The experiment was designed to find out any harmful effect of a medium containing a mixture of PX and aqueous extract of G.glabra on the sperm DNA before and after cryopreservation. Materials and Methods: Fifty mature fertile male mice 8-12 weeks old were used. Epididymal sperms were obtained from caudal region and prepared in vitro by direct activation technique using four media namely; PX alone, G.glabra alone, a mixture of PX with G.glabra and Ham's F-12 (as control medium). Certain sperm function characteristics were examined as well as evaluation of DNA integrity by acridine orange test and comet assay before and after cryopreservation. Results: The results showed a highly significant (P<0.001) improvement in the sperm forward movement, with a highly significant (P<0.001) decrease in DNA abnormality using acridine orange test and comet assay, following in vitro activation by the four media compared to before activation. Following in vitro activation and cryopreservation, the results of epididymal sperm showed a highly significant (P<0.001) decrease in DNA abnormality by using PX medium alone, G.glabra medium alone and the mixture of PX and G.glabra compared to control medium and the results before cryopreservation. Conclusion: According to the results of present study, it has been found no toxic effect of medium containing PX and/or G.glabra on the epididymal sperms DNA after activation in vitro and cryopreservation using acridine orange test and comet assay. The best result obtained when using a medium containing a mixture of PX+ G.glabra together. These data could be applied for preparation and activation of epididymal and testicular sperms obtained from obstructive azoospermic men.