The aim of this study was to observe the effect of selected biochemical parameters of seminal plasma on motility parameters of stallion spermatozoa. Fresh semen was collected from 12 breeding stallions at National Stud Farm in Topoľčianky in age of 5–26 years. Semen was divided in two aliquots. One part of ejaculate was centrifuged (15 minutes at 1000 x g) and the supernatant was separated. The supernatant was frozen and stored at −18°C until analysis. Other part of ejaculate was diluted with physiological solution in ratio 1:3 and stored at 7°C before CASA analysis. Later ejaculates were cultivated at temperature 38°C and total motility, progressive motility and velocity curved line were determined using CASA method at time intervals 0, 1, 2, 3, 6, 24 hours. Assessment of biochemical parameters was realized by Randox RX Monza analyzer. The correlation analysis showed a statistically significant (P<0.05) effect of Mg and Ca on spermatozoa motility. Further, positive significant (P<0.05) correlation was found between Mg and progressive motility. No correlation was found between biochemical parameters of stallion seminal plasma and velocity curved line (VCL). Results indicate that optimal concentration of calcium and magnesium in seminal plasma has beneficial effect on motility parameters of stallion spermatozoa. This phenomenon might be on focus, to improve the percentage of pregnant mares.