The consumption of citric acid, contained in lemon juice, may effectively improve indigestion and maintain an optimal intestinal environment because it promotes gastric acid function. In the present study, we recruited healthy male university students and randomized them into two groups that drank 300 cc of water or lemon water prior to meals for 4 weeks. Stool samples were collected before and after the test period, and the intestinal microbiota was analyzed using next generation sequencing. Bacterial species were identified based on taxonomic classification and binomial nomenclature, and were categorized at the genus level. Bacterial species with a relative abundance rate higher than 0.01% in the water- and lemon water-drinking groups were identified, and changes in the amounts of these bacteria were examined. The results obtained revealed a significant change in the amount of Bilophila, and slight changes in the amounts of Adlercreutzia, Haemophilus, Megamonas, and Roseburia. Therefore, the pre-meal intake of lemon water appeared to promoted peristalsis and digestion, which changed the intestinal microbiota.