This study investigates the diversity and stability of butyrate-producing bacteria in the human gut. Butyrate, a preferred energy source for colonic epithelial cells, plays a crucial role in maintaining colonic health. Anaerobic butyrate-producing bacteria were isolated from fecal samples of three healthy individuals: an infant, an adult omnivore, and an adult vegetarian. The isolates were tested for butyrate production, and 74 isolates produced more than 2 mM butyrate in vitro. These isolates were grouped using 16S ribosomal DNA (rDNA) PCR-restriction fragment length polymorphism (RFLP) analysis. The results showed significant overlap between ribotypes among different individuals and sampling times, indicating substantial diversity and variability. Phylogenetic analysis of 16S rDNA sequences revealed that 80% of the butyrate-producing isolates belonged to cluster XIVa of gram-positive bacteria, with the most abundant group clustering with *Eubacterium rectale*, *Eubacterium ramulus*, and *Roseburia cecicola*. Additionally, 50% of the butyrate-producing isolates were found to consume acetate during growth, suggesting the use of the butyryl coenzyme A-acetyl coenzyme A transferase pathway for butyrate production. The study highlights the importance of understanding the phylogeny and physiology of these bacteria in the context of colonic metabolism and gut health.This study investigates the diversity and stability of butyrate-producing bacteria in the human gut. Butyrate, a preferred energy source for colonic epithelial cells, plays a crucial role in maintaining colonic health. Anaerobic butyrate-producing bacteria were isolated from fecal samples of three healthy individuals: an infant, an adult omnivore, and an adult vegetarian. The isolates were tested for butyrate production, and 74 isolates produced more than 2 mM butyrate in vitro. These isolates were grouped using 16S ribosomal DNA (rDNA) PCR-restriction fragment length polymorphism (RFLP) analysis. The results showed significant overlap between ribotypes among different individuals and sampling times, indicating substantial diversity and variability. Phylogenetic analysis of 16S rDNA sequences revealed that 80% of the butyrate-producing isolates belonged to cluster XIVa of gram-positive bacteria, with the most abundant group clustering with *Eubacterium rectale*, *Eubacterium ramulus*, and *Roseburia cecicola*. Additionally, 50% of the butyrate-producing isolates were found to consume acetate during growth, suggesting the use of the butyryl coenzyme A-acetyl coenzyme A transferase pathway for butyrate production. The study highlights the importance of understanding the phylogeny and physiology of these bacteria in the context of colonic metabolism and gut health.