Semen cryopreservation is a very important technique for assisted reproduction; however, the cryopreservation process is harmful because it results in a reduction in sperm motility and viability, and leads to premature signals of capacitation, resulting in lesser than desirable fertility rates after artificial insemination. A fraction of seminal plasma, enriched in proteins that contain type II fibronectin domains (FNII) can reverse molecular indicators of cryo-capacitation. The beneficial effects of these proteins, however, depend on the relative abundance in seminal plasma. To create a safe additive for improving frozen sperm functionality, in the present study there was cloning and expression of a recombinant peptide containing four FNII domains (named TrxA-FNIIx4-His6) and evaluation of its effect after addition to frozen/thawed ram sperm. The cDNA for this protein was expressed in E. coli and after denaturation and re-naturalization of the protein, toxicity and binding capacity were assessed. By fluorescent labelling assessment, there was binding of the protein to the thawed sperm. At the two doses used (0.15 and 0.3 μM), TrxA-FNIIx4-His6 had the capacity to reverse the molecular indicators of cryo-capacitation as indicated by the reduction on phosphorylated substrates of PKA. Furthermore, the supplementation with this protein resulted in a normal capacitation process as evidenced by the increase in the in vitro fertilization rate when the greatest concentration of the protein was evaluated (73.25 ± 2.95; 40.13 ± 11.82 for 0.3 μM and control, respectively). There was no effect of protein supplementation on sperm objective motility compared to untreated sperm. In conclusion, the use of TrxA-FNIIx4-His6 is a promising biotechnological approach for cryopreserving ram sperm and maintaining sperm viability.