Abstract The objective was to determine the effect of an amino acid-formulated diet on ruminal fermentation in vitro. While most amino acid-formulated diets focus on improving the amino acid composition of rumen undegradable protein, additional benefits to microbial fermentation within the rumen are unknown. The experiment was a completely randomized design with a factorial treatment arrangement (2 donor diets and 2 substrates). Four ruminally cannulated steers were fed one of two donor diets: control (CON; 55% dry rolled corn, 20% corn silage, 15% dry distiller’s grain, and 10% supplement) and amino acid-formulated (AAF; 51% dry rolled corn, 15% corn silage, 14% dry distillers grain, 10% heat-treated soybean meal, and 10% supplement). After a 30-d diet adaptation period, rumen fluid was collected and pooled by donor diet for the in vitro fermentation. Additionally, two different substrates were used to represent each donor diet. Flasks contained 2.5 g dry matter (DM) of substrate and 150 mL of rumen fluid mixture (2 McDougall’s buffer:1 rumen fluid). Treatments were evaluated in triplicate flasks in two replicated runs for a total of six observations per treatment. Four sealed Ankom F57 bags were filled with 0.25 g DM of substrate each to facilitate measuring in vitro DM disappearance (IVDMD). Flasks were incubated at 39°C with sample aliquots collected at h 0, 4, 8, 12, and 24 to determine pH, ammonia, and volatile fatty acids (VFA). Data were analyzed using the MIXED procedure of SAS 9.4 with a repeated measures analysis. Ammonia, pH, and VFA were analyzed with effects of donor diet, substrate, time, and all interactions. A treatment by hour interaction was detected (P < 0.01) for pH; fluid from steers fed CON was greater than AAF at h 4, 8, 12, and 24. A treatment by hour interaction was also detected (P = 0.02) for ammonia; fluid from steers fed CON was greater than AAF at h 24, but was not different at other time points. Total VFA was affected by hour (P < 0.01) as it increased over time, but substrate did not have an effect (P = 0.21). A treatment by hour interaction (P = 0.06) tended to be observed for molar proportions of acetate as AAF and CON were similar at h 0 but AAF was increasing greater than CON over time. A treatment by hour interaction was observed (P = 0.01) for molar proportions of propionate; at h 4 at 8, CON was greater than AAF. No effect (P ≥ 0.32) of donor diet, substrate or their interaction were observed on IVDMD. Overall, an amino acid-formulated diet for cannulated steers did impact ruminal fermentation in vitro, mostly altering molar proportions of volatile fatty acids and ammonia release from protein degradation.