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Purpose: The aim of this study was to evaluate the hypocholesterolemic, immune- and microbiota-modulatory effect of a mushroom extract in hypercholesterolemic subjects., Methods: A randomized, controlled, double-blind, and parallel clinical trial was carried out with subjects from 18 to 65 years old (n = 52) with untreated mild hypercholesterolemia. Volunteers consumed a β-D-glucan-enriched (BGE) mixture (10.4 g/day) obtained from shiitake mushrooms (Lentinula edodes) ensuring a 3.5 g/day of fungal β-D-glucans or a placebo incorporated in three different commercial creams., Results: This mixture showed hypocholesterolemic activities in vitro and in animal studies. After eight weeks intervention, no significant differences in lipid- or cholesterol-related parameters were found compared to placebo subjects as well as before and after the BGE mixture administration. No inflammatory or immunomodulatory responses were noticed and no changes in IL-1β, IL-6, TNF-α or oxLDL were recorded. However, consumption of the BGE mixture was safe and managed to achieve the dietary fibre intake recommended as cardiovascular protective diet. Moreover, the BGE mixture modulated the colonic microbiota differently compared to placebo. Microbial community composition varied from before to after the intervention with several genera being positively or negatively correlated with some biomarkers related to cholesterol metabolism., Conclusion: These results suggested a relation between cholesterol metabolism, microbiota and BGE administration. Nevertheless, the precise significance of this differential modulation was not fully elucidated and requires further studies., (© 2021. Springer-Verlag GmbH, DE part of Springer Nature.)