Large and diverse homoacetogens exist in the gut of herbivores. The aim of this study was to investigate the hydrogenotrophic features of acetogen enrichments (AE) from the gut of cow, sheep, rabbit and horse, and their potential role in converting syngas (H₂ and CO₂) into biofuel products. Rumen contents of 6 late lactation cows and 4 Hu sheep, cecal contents of 6 New Zealand White rabbits, and feces of 4 Australian horses were sampled respectively. Potential acetogens were enriched from each sample using modified anaerobic AC11.1 medium with 2-bromoethanesulfonic acid. Batch-culture experiments using H₂ and CO₂ (4:1) as substrates were conducted to determine the hydrogen consumption and short-chain fatty acids (SCFAs) production of AE. Incubations were lasted for 3 d at 37°C, the ratio of gases in the headspace and concentration of SCFAs in the culture were determined by gas chromatography every 12 h of incubation. Data for the moles of hydrogen consumption and SCFAs production were analyzed by one-way ANOVA in R 3.2 with individual animal as experimental unit, species as main effect. Turkey multiple range tests were used to do multiple comparisons among different groups. Significance was declared if P < 0.05. The maximum cell densities (OD₆₀₀) were observed at 48 h of incubation of all the samples studied, acetate was the major end product and no methane was detected, minor propionate, butyrate and isovalerate were also produced. During 48 h of incubation, AE from horses consumed 4.75 moles of H₂ to 1 mol of acetate, significantly lower to rabbits, cows and sheep (5.17, 5.53 and 5.23 moles respectively), and AE from horses produced significantly more butyrate (0.70 mM) compared with others. AE from cows and sheep produced significantly higher propionate (0.84 mM and 0.86 mM) compared with rabbits and horses (0.69 mM and 0.71 mM). Production of isovalerate was significantly higher of AE from the sheep (0.42 mM). SCFAs are important precursors for the synthesis of various biofuel products, such as polyvinyl acetates, ethanol and butanol et al. In conclusion, the gut contents of herbivores maybe promising resources to harvest functional acetogens for chemical and biofuel production.