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The internal cavity of equine contains symbiotic microorganisms that are collectively referred to as the gut microbiota, which interact with the host immune system from birth. The microorganisms in the gut microbiota are shaped by their interactions with the gut environment throughout the life of the host, i.e., exposure to antibiotics and diet.  Lactobacilli are one of the major groups found in the gastrointestinal tracts of humans and animals. Lactobacilli are members of the lactic acid bacteria, and they help to maintain a balanced gut microbiome and stimulate the host’s immune system. In this study six equine Lactobacillus spp. were isolated from three American Quarter horses of different ages (i.e., 1-day post-weaning, 1.5-month post-weaning, and 10-year-old mature gelding). The metabolic properties that allowed the isolates to survive in the harsh environment of the gut were characterized. Thus, we evaluated their abilities to metabolize different carbohydrates and to withstand acidic pH, bile salts, antibiotics, and to inhibit pathogenic bacteria which may be encountered during their passage to the small/large intestine. We also identified the genetic elements that allow the isolates to survive and persist in the host’s gut environment by using data generated from whole genome sequencing. The data indicated that the isolates were metabolically adapted to the age of the host and the type of feed consumed. The characterized isolates are potential probiotic candidates for enhancing the gut health of equines.