The objective of this study was to examine the effect of partial substitution of soybean meal with a product containing slow-release NPN and microbial fermentation extracts [(OPT); Optimase Alltech Inc., Nicholasville, KY)] on rumen fermentation. This is a companion study to a performance experiment (Díaz-Royón et al., 2014, J. Dairy Sci. 97 (E-Suppl. 1):332). Four cannulated, lactating Holstein cows were randomly assigned to a 4 × 4 Latin square in a 2 × 2 factorial arrangement of treatments. High (HF) and low forage (LF) diets contained, respectively, 61% and 46% forage, with and without OPT. Forages consisted of 75% corn silage and 25% alfalfa hay (DM basis). Experimental diets containing OPT (125 g/cow/day) were designed to partially replace soybean meal 48% CP (0.625 kg) with forage fiber [corn silage (0.225 kg) and alfalfa hay (0.075 kg)] and non-forage fiber [soybean hulls (0.200 kg)]. Increasing forage produced the expected pH increase (6.15 vs. 6.05 for HF and LF, respectively; P < 0.01) whereas ruminal pH was not affected by OPT inclusion. Ruminal ammonia concentration tended to be greater in cows fed HF than LF diets (8.07 vs. 7.28 mg/dL; P = 0.08), whereas OPT had no effect. Cows fed HF diets had a lower total VFA concentration compared with those fed LF (86.5 vs. 91.6 mM; P = 0.02). For cows fed HF diets, molar proportions of acetate (66.0 vs. 64.4%; P < 0.01), isobutyrate (0.89 vs. 0.82%; P < 0.01), and isovalerate (1.80 vs. 1.58%; P < 0.01) were greater, whereas those for propionate were lesser (19.4 vs. 21.1%; P < 0.01). Total VFA proportions increased by 6.4% in HF diets supplemented with OPT; however, it decreased by 4.4% on LF diets (forage × OPT, P value = 0.03). Feeding OPT increased molar proportions of acetate (P < 0.01), decreased propionate (P < 0.01), isobutyrate (P < 0.01), and isovalerate (P < 0.01), but did not change molar proportions for butyrate and valerate. Partial replacement of soybean meal with OPT in dairy cow diets altered ruminal fermentation in a manner that suggests improved fiber digestion.