Chemical elements, as structural components of the body, influence physiological processes and functions of the organism, growth, productive and reproductive traits, resistance and others. In this regard, the present study aimed to evaluate the effect of different calcium concentrations in the wool of Holstein sires on the semen quality characteristics. The studies were conducted in conditions of Leningrad and Vologda regions on Holstein sires, 3–5 years old (n = 40). Calcium concentration in wool was determined by atomic emission and mass spectrometry (AES–ICP and MS–ICP). According to the results of this analysis and comparison with previously developed reference intervals, we formed four groups of sires: I – level of Ca concentration less than 25th percentile, II and IV – within 25–75th percentile (physiological norm: 1597–2926 mg/kg) and III – more than 75th percentile. As a result of this distribution, the Ca concentration in the wool of animals of Group II was higher by 75.9 % compared to Group I; in turn, individuals of Group IV exceeded Group III – by 27.9 %. According to the results of semen quality characteristics evaluation, it was found that in the group of sires with Ca level within the previously developed reference intervals, the concentration of spermatozoa was higher by 15.3 % and their activity – by 0.17 points in fresh semen in comparison with the group with its reduced level (below the 25th percentile). High Ca content (above the 75th percentile) in wool contributed to a 9.0 % decrease in the concentration of spermatozoa compared to its average values. Thus, the calcium concentration, assessed by its level in the fur, affects the concentration and activity of sperm. It is necessary to evaluate it to improve the quality characteristics of sperm systematically.