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Gut microbiota alterations are intimately linked to chronic constipation upon aging. We investigated the role of targeted changes in the gut microbiota composition in the relief of constipation symptoms after rhubarb extract (RE) supplementation in middle-aged volunteers. Subjects (95% women, average 58 years old) were randomized to three groups treated with RE at two different doses determined by its content of rhein (supplementation of 12.5 mg and 25 mg per day) vs. placebo (maltodextrin) for 30 days. We demonstrated that daily oral supplementation of RE for 30 days was safe even at the higher dose. Stool frequency and consistency, and perceived change in transit problem, transit speed and difficulty in evacuating, investigated by validated questionnaires, were improved in both groups of RE-treated volunteers compared to placebo. Higher abundance of <i>Lachnospiraceae</i> (mainly <i>Roseburia</i> and <i>Agathobacter</i>) only occurred after RE treatment when present at low levels at baseline, whereas an opposite shift in short-chain fatty acid (SCFA) levels was observed in both RE-treated groups (increase) and placebo (decrease). Fecal <i>Lachnospiraceae</i> and SCFA were positively correlated with stool consistency. This study demonstrates that RE supplementation promotes butyrate-producing bacteria and SCFA, an effect that could contribute to relieving chronic constipation in middle-aged persons.