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Abstract Due to the preventive effect that plant sterols could have in relation to colon cancer and the scarce information available on plant sterols-gut microbiota interaction, we evaluate the sterols influence upon gut microbiota and viceversa. In vitro colonic fermentation using a residue from the in vitro digestion of a plant sterol-enriched beverage were used. Faecal sterols by GC–MS, and gut microbiota using DNA sequencing were determined. A higher plant sterols metabolism and lower for cholesterol in presence of plant sterols was occurred. Neutral plant sterols decreased and its metabolites increased during fermentation times. The global changes in microbial communities were associated to fermentation time regardless the sterol supplementation. Notwithstanding, plant sterols decreased the proportion of Erysipelotrichaceae species and increased the abundance of phylotypes associated with Eubacterium hallii. The study confirms a higher plant sterols metabolism against cholesterol by gut microbiota. Plant sterols could help to increase the beneficial species abundance.