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The experiment was conducted to evaluate the effect of yeast culture (Saccharomyces cerevisiae) supplementation on ruminal fermentation in 20 Holstein dairy cows divided into control and experimental groups, each group of 10 cows. The animals received a diet based on maize silage (19 kg), alfalfa silage (15 kg), meadow hay (1.5 kg), extracted rapeseed meal (1 kg) and concentrate mixture (9.5 kg). The diets were fed as a total mixed ration. The supplement of yeast culture Levucell® SC 20 (Saccharomyces cerevisiae - CNCM I-1077; min. content 2 x 1010 CFU⋅g-1) was added to the concentrate mixture in the ration fed to the experimental group of animals. The addition of yeast culture significantly (P < 0.01) increased ruminal pH but had no positive effects on the increased production of volatile fatty acids. The supplementation of yeast culture significantly (P < 0.01) increased numbers of protozoa in the rumen of dairy cows of the experimental group (361.3 ± 18.315) compared to the control group (308.3 ± 37.505). The addition of yeast culture significantly (P < 0.01) increased concentration of serum glucose, calcium, phosphorus, copper, zinc, magnesium and AST (P < 0.05). As compared to the control group (4.948 ± 0.0384 mmol⋅1-1), the level of urea in the blood serum was significantly decreased (P < 0.01) in the experimental group of cows. Our results show that the supplementation of Saccharomyces cerevisiae culture at recommended doses enhances ruminal fermentation which may have a positive effect on milk production and health status of Holstein dairy cows. [ABSTRACT FROM AUTHOR]