Background: Probiotics have been observed to positively influence the host's health, but to date few data about the ability of probiotics to modify the gut microbiota composition exist. Aims: To evaluate the ability of Lactobacillus kefiri LKF01 DSM32079 (LKEF) to colonize the intestinal environment of healthy subjects and modify the gut microbiota composition. Methods: Twenty Italian healthy volunteers were randomized in pre-prandial and post-prandial groups. Changes in the gut microbiota composition were detected by using a Next Generation Sequencing technology (Ion Torrent Personal Genome Machine). Results: L. kefiri was recovered in the feces of all volunteers after one month of probiotic administration, while it was detected only in three subjects belonging to the pre-prandial group and in two subjects belonging to the post-prandial group one month after the end of probiotic consumption. After one month of probiotic oral intake we observed a reduction of Bilophila, Butyricicomonas, Flavonifractor, Oscillibacter and Prevotella. Interestingly, after the end of probiotic administration Bacteroides, Barnesiella, Butyricicomonas, Clostridium, Haemophilus, Oscillibacter, Salmonella, Streptococcus, Subdoligranolum, and Veillonella were significantly reduced if compared to baseline samples. Conclusion: L. kefiri LKF01 showed a strong ability to modulate the gut microbiota composition, leading to a significant reduction of several bacterial genera directly involved in the onset of pro-inflammatory response and gastrointestinal diseases. (c) 2016 The Authors. Published by Elsevier Ltd on behalf of Editrice Gastroenterologica Italiana S.r.l. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/bync- nd/4.0/).