The objective of this study was to evaluate the effect of a commercial yeast culture, [Cellerate Culture Classic (CC), Phibro Animal Health Corp.] on volatile fatty acid (VFA) production 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 (0.0567 g/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 and ammonia nitrogen. Three replicate incubation days were performed and data were analyzed using PROC GLIMMIX of SAS; significance was defined as P ≤ 0.05. Treatment did not affect gas production kinetics, final pH, final temperature, ammonia nitrogen, isobutyric acid (mmol/L), acetate: propionate, or acetate, propionate, isobutyrate, isovalerate, or valerate proportions (percent of total VFA). CC (vs CNTL) increased total acetate (72.67 vs 68.12 ± 4.69 mmol/L; P = 0.01), propionate (24.02 vs 22.21 ± 2.68 mmol/L; P = 0.003), butyrate (10.22 vs 9.32 ± 0.68 mmol/ L; P = 0.001), isovalerate (0.83 vs 0.76 ± 0.02 mmol/L; P = 0.01), valerate (1.51 vs 1.40 ± 0.32 mmol/L; P = 0.006) and total VFA concentrations (110.17 vs 102.69 ± 8.22 mmol/L; P = 0.005). CC increased butyrate proportions vs CNTL (P = 0.001). Our results indicate that adding yeast culture to a lactating cow diet may increase volatile fatty acid production without altering kinetics of gas production, pH, or temperature in an in vitro system which may modulate ruminal fermentation.