The effect of steroid hormones on the quality of fresh and cryopreserve sperm has not been fully understood. This study aimed to evaluate the effect of testosterone, dihydrotestosterone, estradiol, and cortisol on the quality of fresh and cryopreserved stallion sperm. The study was conducted on 40 Equus caballus stallions, including Arab (n=20), Oryol trotting (n=4), Standardbred (n=4), and Soviet Heavy Draft (n=12) breeds. The average age of the experimental animals was 9.9 ± 0.7 years. We determined standard quality indicators in fresh and cryopreserved sperm and the concentration of steroid hormones in the blood plasma of stallions. Results of the study suggested a negative correlation between the level of testosterone with total (r=-0.41; p<0.01) and progressive (r=-0.44; p<0.01) sperm motility in cryopreserved sperm as well as in fresh sperm (r=-0.38; p<0.05 and r=-0.39; p<0.05 correspondingly). While the level of estradiol showed a positive correlation with survival rate in cryopreserved (r=0.35; p<0.05) and in fresh (r=0.33; p<0.05) sperm. Further, the level of cortisol in the blood plasma of stallions did not show any statistically significant correlations with the qualitative characteristics of sperm.  A positive relationship was found between the concentration of dihydrotestosterone with the volume of ejaculate (r=0.37; p<0.05) and the total number of sperm in the ejaculate (r=0.43; p<0.01). Results of the study can be concluded that steroid hormones have different effects on the quality indicators of fresh and cryopreserved sperm of stallions and their concentration in the blood should be considered when selecting stallions for cryopreservation of sperm.