Heat stress (HS) and dietary crude protein (CP) affect dairy cattle production through alterations of rumen microbial fermentation. The objective of this study was to determine in vitro fermentability, pH, and volatile fatty acid and gas production of rumen content from donor cows exposed to environmental and dietary treatments. A completely randomized design with a 2 × 2 factorial arrangement of treatments was conducted with 12 multiparous Holstein cows (215 ± 8 d in milk) for 21 d. Donor cows received treatments that consisted of HS or cooling with fans and sprinklers (CO) and high (17.9%, HCP) or low (14.2%, LCP) CP diets on a DM basis. Cows were exposed to prevailing July and August weather in Tennessee. Rumen fluid was collected by stomach tubing from each animal (3 cows/treatment) to determine individual in vitro fermentability. Data presented herein were determined at the end of 48 h in vitro incubation (ANKOM Technology, Macedon, NY). Metabolite concentrations were measured using HPLC, ¹H nuclear magnetic resonance, and ion chromatography. Treatment effects were tested using the Mixed procedure in SAS (LSM ± SEM). In vitro concentrations of volatile fatty acids did not change. At 48 h in vitro incubation, there was an interaction (P < 0.01) showing a greater reduction in gas production from LCP vs. HCP diet under CO (96.8 vs. 63.7 ± 10.9 mL/g of DM) relative to that under the HS treatment (19.2 vs. 17.7 ± 10.9 mL/g of DM). There was an interaction (P = 0.04) showing a greater reduction of ammonium concentrations in vitro from LCP vs. HCP diet under HS (12.7 vs. 1.45 ± 1.19 mM) relative to that under the CO treatment (10.4 vs. 4.17 ± 1.19 mM). Fluid incubation from HS cows increased (P < 0.01) rumen pH compared with that from CO cows (6.99 vs. 7.15 ± 0.03). Compared with HCP, LCP fluid showed an increase (P = 0.02) in pH (7.02 vs. 7.12 ± 0.03) and a decrease (P < 0.01) in methane concentration (20.9 vs. 15.4 ± 1.20 g/mL). In summary, results revealed that rumen content from cows exposed to HS and a low CP diet affected metabolite production in vitro.