The effect of a Citrus Fruit Extract high in the polyphenols hesperidin and naringin (CFE) on modulation of the composition and activity of the gut microbiota was tested in a validated, dynamic in vitro model of the colon (TIM-2). CFE was provided at two doses (250 and 350 mg/day) for 3 days. CFE led to a dose-dependent increase in Roseburia, Eubacterium ramulus, and Bacteroides eggerthii. There was a shift in production of short-chain fatty acids, where acetate production increased on CFE, while butyrate decreased. In overweight and obesity, acetate has been shown to increase fat oxidation when produced in the distal gut, and stimulate secretion of appetite-suppressive neuropeptides. Thus, the data in the in vitro model point towards mechanisms underlying the effects of the polyphenols in CFE with respect to modulation of the gut microbiota, both in composition and activity. These results should be confirmed in a clinical trial.