Abstract This study compared the effects of feeding a new strain of Saccharomyces cerevisiae HSA2020 with a commercial strain on in vitro rumen fermentation and production performance of dairy cows. Permeate was used as a substrate for the laboratory production of the new strain of S. cerevisiae after the hydrolysis by β-galactosidase (5000 µ/mL at 37°C). Two experiments were conducted: in Experiment 1, the effects of three levels (1, 2 and 3 g/kg dry matter) of S. cerevisiae on in vitro ruminal fermentation kinetics were evaluated. In Experiment 2, for 60 days, sixty multiparous Holstein cows (639±24.8 kg BW, 3±1 parity, 7±1 days in milk, with a previous milk production of 23±2.0 kg/d) during the previous lactation, were randomly assigned to 3 treatments in a completely randomized design. Cows were fed without any additives (control treatment) or supplemented with 2 g/kg feed daily of laboratory produced (PY) or commercial (CY) S. cerevisiae. In Experiment 1, inclusion of PY and CY increased (P<0.05) gas production, propionate, and nutrient disappearance, while decreased (P<0.05) methane production and protozoal count. Moreover, in Experiment 2, PY followed by CY increased (P<0.01) nutrient digestibility, and serum concentrations of total protein, albumin, and glucose (P<0.05). Higher daily milk yield, and milk energy output were observed with PY and CY without affecting concentrations of milk components or milk fatty acid profile. Compared to control, increased feed efficiency was observed with PY and CY. Compared to PY, CY increased serum concentrations of urea-N and decreased triglycerides, while PY decreased serum aspartate transaminase and increased concentration of conjugated linoleic acids in milk. In early lactating cow diets, both strains of S. cerevisiae improved production performance at 2 g/kg, and minimal differences between strains were found.