High-yield dairy cows with high-concentrate diets are more prone to experiencing health problems associated with rumen microbial imbalance. This study assessed the effects of Saccharomyces cerevisiae culture (SC), a food supplement, on ruminal pH, volatile fatty acid (VFA), inflammatory cytokines, and performance of high-yield dairy cows. Forty Holstein cows with similar characteristics (e.g., milk yield, days of milk, and parity) were randomly divided into two groups: an experimental group fed the basal ration supplemented with the SC of 100 g of SC per cow per day (hour, SC group), and a control group fed the same basal ration diet without SC (i.e., CON group). On average, the supplementation of SC started at 73 days of lactation. The experimental period lasted approximately 70 days (from 18 January to 27 March 2020), including 10 days for dietary adaptation. Milk yield was recorded daily. Rumen fluid and milk samples were collected after 2 h of feeding in the morning of day 0, 15, 30, and 60. The data showed that rumen pH increased (p < 0.05) when cows were provided with SC. On average, the cows in the SC group produced 1.36 kg (p < 0.05) more milk per day than those in the CON group. Milk fat content of cows in the SC group was also higher (4.11% vs. 3.96%) (p < 0.05). Compared with the CON group, the concentration of acetic acid in the rumen fluid of dairy cows in the SC group was significantly higher (p < 0.05). There were no differences (p > 0.05) found in milk protein content and propionic acid between groups. The SC group had a tendency increase in butyric acid (p = 0.062) and total VFA (p = 0.058). The result showed that SC supplementation also enhanced the ratio between acetic and propionic. Most of the mean inflammatory cytokine (IL-2, IL-6, γ-IFN, and TNF-α) concentrations (p < 0.05) of the SC group were lower than CON group. This study demonstrated that high-yield cows receiving supplemental SC could produce more milk with higher fat content, have higher rumen acetate, and potentially less inflammatory cytokines.