High starch diets are known to reduce rumen pH, and cause shifts in the VFA profile, resulting in potential health problems. The aim of this study was to investigate if the addition of autolyzed yeast (AY; Saccharomyces cerevisiae) supplementation in a high starch lactation diet would improve rumen pH, fecal pH, and shifts in VFA response. Fifteen rumen-cannulated Holstein cows were assigned to 1 of 5 treatments in a replicated 5 × 5 Latin square design balanced to measure carryover effects. Treatments were: low starch diet without AY (LS0; control), high starch diet without AY (HS0), high starch diet with either 15 g (HS15), 30 g (HS30), or 45 g (HS45) of AY supplementation. The period of 21 d was divided into the adaptation phase (d 1 to 14) and a measurement phase (d 15 to 21). Rumen fluid was collected via rumen cannula on d 15 and 16 in relation to feeding at 1400 h (time point 0). Rumen samples were extracted at 0, 4, 8, 12, 16, 20, and 24 h relative to feeding. Cows in HS0 experienced lower ruminal pH (6.10 vs. 6.38; P < 0.0001), nadir pH (5.53 vs. 5.74; P < 0.0001), and fecal pH (6.71 vs. 6.95; P = 0.042) compared with LS0. The addition of AY increased rumen pH (P = 0.04), and nadir pH (P = 0.009), compared with HS0 with no effect on fecal pH. Supplementing AY reduced individual VFA proportions of acetate, isobutyrate, and isovalerate (P = 0.02;P = 0.0004 and P = 0.002, respectively) when compared with cows in HS0. Total VFA proportion was greatest (136.71mmol/L; P = 0.0005) in cows fed HS0 compared with LS0. Total VFA proportions were greater for propionate (23.87 vs. 20.75%; P < 0.0001) and valerate (1.50 vs.1.35%; P = 0.0001) for cows in HS0 than LS0. Supplementing AY positively increased total propionate proportion (P = 0.002) and negatively decreased total acetate (P < 0.0001), isobutyrate, (P = 0.0003), and isovalerate (P = 0.01) when compared with HS0. Total VFA acetate (65.51 vs. 62.41%; P < 0.0001), isobutyrate (0.85 vs. 0.78%; P = 0.0001), and isovalerate (0.63 vs. 0.60%; P = 0.02) were greater in LS0 treatment compared with HS0. In conclusion, these results confirm the addition of AY aids in increased rumen pH values and shifts in VFA response.