The objective of this study was to evaluate the effects of supplementing a yeast culture (YC; Saccharomyces cerevisiae) on rumen fermentation, nutrient utilization, and milk production in dairy cows. Six Holstein cows were subjected to two dietary treatments in a crossover design. The treatments were control (no yeast culture) and yeast culture (fed at 30 g/head per day). Rumen pH and protozoal counts were similar between both treatments, although the concentration of rumen ammonia-N tended to be lower with YC treatment. Moreover, YC treatment did not affect the concentration of total short-chain fatty acids in the rumen, but tended to reduce the molar proportion of acetic acid (A), while significantly increasing that of propionic acid (P). Therefore, the A/P ratio decreased significantly after 5 hr of YC feeding as compared with the control. The treatments had no effect on the blood plasma concentrations of glucose, urea-N, and non-esterified fatty acids. However, YC treatment resulted in a significantly higher concentration of plasma free lysine, along with higher concentrations of isoleucine, arginine, and total free amino acids. Dry matter intake and milk yield were similar between both treatments, though the percentage of milk protein tended to be higher with YC treatment. In addition, YC treatment did not affect the milk fatty acid composition and somatic cell counts. Overall, the yeast culture had a subtle effect on ruminal fermentation. The higher plasma concentrations of some essential amino acids indicated the positive effect of YC supplementation on the availability of amino acids for milk protein synthesis in the mammary gland of dairy cows.