This study was conducted to determine the nutritive value of live yeast culture (RumiSacc, Saccharomyces cerevisiae) and to investigate its effects on milk yield, milk composition and some blood parameters in lactating cows. Six multiparous Holstein cows were allocated to two groups of three cows and assigned randomly to one of two diets in a cross-over experiment. Daily 50 g RumiSacc was top dressed at the p.m. feeding for the treatment group. RumiSacc supplied a high protein and energy with high organic matter digestibility values (83.35%) determined by in vitro enzymatic analysis. Yeast culture supplementation significantly increased milk yield, tended to increase fat yield, protein yield and lactose yield of milk. Methylated fatty acid level of 18:3 (n-3) in milk fat was increased by yeast culture supplementation. The concentrations of methionine, phenyalanine, tyrosine, tryptophan and taurine were significantly increased with dietary inclusion of yeast culture. Live yeast culture supplementation did not affect other performance characteristics, milk quality characteristics and blood parameters. As a conclusion live yeast culture (RumiSacc, Saccharomyces cerevisiae) had high nutritive value and positive effects on milk production and some milk quality characteristics in lactating cows under field conditions.