The sensitivity of ram semen to cryobiological procedures has motivated many researchers to investigate this issue. In clinical practice, one of the main objectives is to reduce the damage caused by freezing cells through the addition of preservatives to semen diluents. In our study, fetal calf serum (15% FCS) and 3–6 mM cysteamine were added to ram semen diluents, and their effects on semen after freeze-thawing were investigated. Semen was collected from five Kıvırcık rams using an electroejaculator and combined in equal volumes (pooling). After macroscopic and microscopic examination, the samples were divided into six equal volumes. Six study groups were established by adding 15% FCS, 3 mM cysteamine, 3 mM cysteamine + 15% FCS, 6 mM cysteamine, and 6 mM cysteamine + 15% FCS to the second, third, fourth, fifth, and sixth aliquots, respectively. The first group was maintained for control purposes. The diluted semen was drawn into 0.25 mL straws after equilibration, frozen at –110 °C, and stored at 196 °C. After equilibration and thawing, semen was evaluated using phase-contrast microscopy, computer-assisted semen analysis (CASA), the hypoosmotic swelling test (HOST), and the Hancock method. Motility decreased, and the number of dead spermatozoa increased in the groups treated with 6 mM cysteamine (Groups 5 and 6), whereas there were no significant changes in spermatozoa motility or morphology in the other groups (control group and Groups 2, 3, and 4). In conclusion, 3 mM cysteamine and FCS (15%) had a statistically significant synergistic effect on spermatozoa motility after equilibration (p < 0.05), yet this effect was not observed after freeze-thawing.