Background: Numerous studies have investigated the effects of the supplementation of fructooligosaccharides (FOS) on the number of bacteria in the gut that are good for health, but the results have been inconsistent. Additionally, due to its high fermentability, supplementation of FOS may be associated with adverse gastrointestinal symptoms such as bloating and flatulence. Therefore, we assessed the effects of FOS interventions on the composition of gut microbiota and gastrointestinal symptoms in a systematic review and meta-analysis. Design: All randomized controlled trials published before 10 July 2022 that investigated the effects of FOS supplementation on the human gut microbiota composition and gastrointestinal symptoms and met the selection criteria were included in this study. Using fixed or random-effects models, the means and standard deviations of the differences between the two groups before and after the intervention were combined into weighted mean differences using 95% confidence intervals (CIs). Results: Eight studies containing 213 FOS supplements and 175 controls remained in this meta-analysis. Bifidobacterium spp. counts significantly increased during FOS ingestion (0.579, 95% CI: 0.444−0.714) in comparison with that of the control group. Subgroup analysis showed greater variation in Bifidobacterium spp. in adults (0.861, 95% CI: 0.614−1.108) than in infants (0.458, 95% CI: 0.297−0.619). The increase in Bifidobacterium spp. counts were greater in the group with an intervention duration greater than 4 weeks (0.841, 95% CI: 0.436−1.247) than an intervention time less than or equal to four weeks (0.532, 95% CI: 0.370−0.694), and in the group with intervention doses > 5 g (1.116, 95% CI: 0.685−1.546) the counts were higher than those with doses ≤ 5 g (0.521, 95% CI: 0.379−0.663). No differences in effect were found between FOS intervention and comparators in regard to the abundance of other prespecified bacteria or adverse gastrointestinal symptoms. Conclusions: This is the first meta-analysis to explore the effect of FOS on gut microbiota and to evaluate the adverse effects of FOS intake on the gastrointestinal tract. FOS supplementation could increase the number of colonic Bifidobacterium spp. while higher dose (7.5−15 g/d) and longer duration (>4 weeks) showed more distinct effects and was well tolerated.