The objective of this study was to evaluate the effect of a commercial yeast culture, [Cellerate Culture Classic Plus (CC+), Phibro Animal Health Corp.] on volatile fatty acid (VFA) production, in vitro dry matter digestibility (IVDMD), and kinetics of gas production using an in vitro rumen fermentation system and lactating cow TMR. Bottle (250 mL) was considered the experimental unit; each bottle contained TMR (1.4 g) and treatment in 100 mL of buffered rumen fluid. Treatments were: CNTL (no additive) and CC+ (14 mg /bottle). All treatments were incubated in triplicate for 24 h at 39°C and constant agitation (60 rpm). Bottles were capped with units to capture temperature and pressure every 15 min (RF1, Ankom Technology, Macedon, NY). At the end of incubation, final pH and temperature measurements were taken; a sample of rumen fluid was frozen for analysis of VFA. Triplicate 50-mL centrifuge tubes were incubated in parallel with larger bottles to asses IVDMD; tubes contained 0.5 g TMR, 5 mg CC+ and 32 mL buffered rumen fluid. Three replicate incubation days were performed, data were analyzed using PROC GLIMMIX of SAS; significance was defined as P ≤ 0.05 and tendencies as 0.05 < P ≤ 0.10. Treatment did not affect gas production kinetics, final pH, final temperature, IVDMD, isobutyric, isovaleric, or valeric acid (mmol/L) concentrations, or VFA production analyzed on a proportional basis. Including CC+ (vs CNTL) increased total acetic acid (70.09 vs 68.12 ± 4.01 mmol/L; P = 0.02), butyric acid (9.51 vs 9.32 ± 0.56 mmol/L; P = 0.04) and total VFA concentrations (105.38 vs 102.69 ± 7.12 mmol/L; P = 0.02). Our results indicate adding yeast culture to a lactating cow diet may increase VFA production without altering kinetics of gas production, pH or temperature in an in vitro system which may modulate ruminal fermentation.