With the aim of finding an ideal cryoprotectant in a suitable concentration for red deer epididymal spermatozoa cryopreservation, we evaluated the effects of three most commonly used cryoprotectants (CPAs), Glycerol (GLY), Ethylene glycol (EG) and Propylene glycol (PG), on sperm cryoresistance. The aim of Experiment 1 was to evaluate the influence of 3 different final concentrations (3, 6 and 12%) of each CPAs on sperm freezability. Sperm samples were diluted to a final sperm concentration of ~400 x 106 spermatozoa/mL with a Tris-citrate-fructose-EY extender (TCF) prior to freezing. Sperm cryosurvival was judged in vitro by microscopic assessments of individual sperm motility (SMI), viability and of plasma membrane (by means of the HOS test) and acrosome (NAR) integrities. Thawed samples were incubated at 37 °C for 2 h in the freezing medium. At the end of this incubation period, sperm suspensions were again assessed. Our results showed that 12% of any CPA was toxic to red deer epididymal spermatozoa membrane integrity (P < 0.05). Moreover, regardless the level of CPA, results indicated that the cryoprotective effects to red deer epididymal spermatozoa of the three CPAs after thawing are in the following sequence: GLY > EG > PG (higher symbols mean P < 0.001). Furthermore, our results also showed an improvement on sperm parameters when the TCF diluent contained 6% of GLY. These results were statistically significant (P < 0.05) at thawing and after 2 h of incubation at 37 °C. In Experiment 1, spermatozoa frozen in diluents containing glycerol (especially 6%) had significantly higher post-thaw viability than those frozen in diluents containing others CPAs. Therefore, in Experiment 2 extenders were prepared using GLY 6%. This experiment was designed to investigate how different temperatures of GLY addition - 22°C (ambient temperature) and 5°C- affected the post-thaw quality of cryopreserved red deer epididymal spermatozoa. Our results showed a differential response (P < 0.05) of motility (SMI) to temperature of GLY addition before freezing, the best being 22°C (81.94 ± 2.4% vs 72.38 ± 2.4%). Although, there were no statistically significant differences (P > 0.05) between the two temperatures of glycerol addition after thawing on sperm quality, after two hours of incubation, results tended to be better when CPAs were added at 22°C. In conclusion, our work showed the efficacy of a TCF diluent with 6% of glycerol and its addition at 22°C, as an alternative to the more common 3-4% of glycerol and addition at 5°C, in red deer semen freezing protocols.