This study was aimed at determining the effects of propionic acid supplementation at doses of 0 (control group, PAO), 12, 24, 48 and 96 mM (PA12, PA24, PA48, and PA96) to concentrated mix feed on in vitro cumulative total gas production, methane emission, gas kinetics (potential gas production, (a + b)(gas) and gas production rate, c(gas)), estimated digestibility, estimated energy value and the end-products and variables of in vitro digestion (total bacteria count, the number of ciliate protozoa, volatile fatty acids, pH value and ammonia-N). Digestion treatments were carried out in an anaerobic in vitro fermenter for up to 96 h. The in vitro cumulative total gas production, (a + b)(gas), estimated metabolic energy, estimated net energy lactation and estimated organic matter digestibility and ammonia-N concentration were decreased by propionic acid up to 96 mM (P < 0.05). In the in vitro fermenter fluid, total bacteria count, the total numbers of ciliate protozoa and the individual numbers of some ciliate protozoa (Entodiniinae, Isotricha spp. and Diplodiniinae) (P < 0.01) decreased linearly with increasing concentrations of dietary propionic acid. The total molar concentrations of volatile fatty acids decreased in response to propionic acid supplementation (P < 0.001). Dietary propionic acid elicited linear increases in the molar concentrations of propionic acid (P < 0.001) and butyric acid (P < 0.01) as proportions of total volatile fatty acids of the in vitro fermenter fluid. In contrast, molar proportions of acetic acid, the c(gas), pH values and the numbers of Dasytricha sp. were not affected by dietary propionic acid supplementation (P > 0.05). The addition of 12-96 mM propionic acid to concentrated mix feed decreased methane emission from the rumen and negatively affected microbiota count, feed digestibility, proteolysis, and molar volatile fatty acid values in the rumen environment.