INTRODUCTION: Gut microbiota manipulation may be a potential therapeutic target to reduce host energy storage. There is limited information about the effects of probiotics/synbiotics on intestinal microbiota composition in children and adolescents with obesity. The objective of this randomized double-blind placebo-controlled trial was to test the effects of a multispecies synbiotic on intestinal microbiota composition in children and adolescents with exogenous obesity. METHOD: Children with exogenous obesity were managed with a standard diet and increased physical activity and were randomly allocated into two groups at a ratio of 1:1; the 1st group received synbiotic supplementation (probiotic mixture including Lactobacillus acidophilus, Lacticaseibacillus. rhamnosus, Bifidobacterium bifidum, Bifidobacterium longum, Enterococcus faecium (total 2.5 × 10 CFU/sachet) and fructo-oligosaccharides (FOS; 625 mg/sachet) for 12 weeks; the 2nd group received placebo once daily for 12 weeks. Fecal samples were obtained before and at the end of the 12-week intervention to characterize the changes in the gut microbiota composition. Detailed metagenomic and bioinformatics analyses were performed. RESULTS: Before the intervention, there were no significant differences in alpha diversity indicators between the synbiotic and placebo groups. After 12 weeks of intervention, the observed taxonomic units and Chao 1 were lower in the synbiotic group than at baseline (p < 0.001 for both). No difference for alpha diversity indicators was observed in the placebo group between baseline and 12 weeks of intervention. At the phylum level, the intestinal microbiota composition of the study groups was similar at baseline. The major phyla in the synbiotic group were Firmicutes (66.7%) and Bacteroidetes (18.8%). In the synbiotic group, the Bacteroidetes phylum was higher after 12 weeks than at baseline (24.0% vs. 18.8%, p < 0.01). In the synbiotic group, the Firmicutes/Bacteroidetes ratio was 3.54 at baseline and 2.75 at 12 weeks of intervention (p < 0.05). In the placebo group, the Firmicutes/Bacteroidetes ratio was 4.70 at baseline and 3.54 at 12 weeks of intervention (p < 0.05). After 12 weeks of intervention, the Firmicutes/Bacteroidetes ratio was also lower in the synbiotic group than in the placebo group (p < 0.05). In the synbiotic group, compared with the baseline, we observed a statistically significant increase in the genera Prevotella (5.28-14.4%, p < 0.001) and Dialister (9.68-13.4%; p < 0.05). Compared to baseline, we observed a statistically significant increase in the genera Prevotella (6.4-12.4%, p < 0.01) and Oscillospira (4.95% vs. 5.70%, p < 0.001) in the placebo group. In the synbiotic group, at the end of the intervention, an increase in Prevotella, Coprococcus, Lachnospiraceae (at the genus level) and Prevotella copri, Coprococcus eutactus, Ruminococcus spp. at the species level compared to baseline (predominance of Eubacterium dolichum, Lactobacillus ruminis, Clostridium ramosum, Bulleidia moorei) was observed. At the end of the 12th week of the study, when the synbiotic and placebo groups were compared, Bacteroides eggerthi species were dominant in the placebo group, while Collinsella stercoris species were dominant in the synbiotic group. CONCLUSION: This study is the first pediatric obesity study to show that a synbiotic treatment is associated with both changes intestinal microbiota composition and decreases in BMI. Trial identifier: NCT05162209 (www. CLINICALTRIALS: gov).