This study aimed to determine the effect of 0 (FA0) and 1, 2, 4 or 8 mL/kg (FA1, FA2, FA4 and FA8) formic acid addition to barley-based concentrated mix feed on in vitro gas kinetics [gas production from quickly soluble fraction (a(gas)), gas production constant rate (c(gas)), gas production of insoluble fraction (b(gas)), potential gas production (a+b)(gas)], methane production, organic matter digestibility (OMD), metabolic energy (ME), net energy lactation (NEL), pH, ammonia-N, volatile fatty acids (VFAs), total bacteria count and number of ciliate protozoa. The in vitro cumulative gas production, c(gas), b(gas), (a+b)(gas), ME, NEL and OMD values and ammonia-N concentration were decreased by formic acid (P < .05). Formic acid supplementation increased in vitro methane production up to about 12% (P < .05). Total bacteria count and number of protozoa and molar concentrations of total VFAs, acetic, propionic and butyric acids decreased with formic acid supplementation (P < .001). The numbers of Isotricha spp. and Dasytricha spp. increased with high formic acid (P < .001). The numbers of Diplodiniinae and Entodiniinae decreased in FA4 and FA8 groups (P < .05). The results indicated that formic acid addition to feed decreased ruminal microbiota count, digestibility, proteolysis and molar VFA values also increased ruminal methane emission. Besides, formic acid supplementation could increase energy loss during ruminal fermentation of feed.