AbstractBlueberries are a rich source of polyphenols, widely studied for the prevention or attenuation of metabolic diseases. However, the health contribution and mechanisms of action of polyphenols depend on their type and structure. Here, we evaluated the effects of a wild blueberry polyphenolic extract (WBE) (Vaccinium angustifolium Aiton) on cardiometabolic parameters, gut microbiota composition and gut epithelium histology of high-fat high-sucrose (HFHS) diet-induced obese mice and determined which constitutive polyphenolic fractions (BPF) was responsible for the observed effects. To do so, the whole extract was separated in three fractions, F1) Anthocyanins and phenolic acids, F2) oligomeric proanthocyanidins (PACs), phenolic acids and flavonols (PACs degree of polymerization DP < 4), and F3) PACs polymers (PACs DP > 4) and supplied at their respective concentration in the whole extract. After 8 weeks, WBE reduced OGTT AUC by 18.3% compared to the HFHS treated rodents and the F3 fraction contributed the most to this effect. The anthocyanin rich F1 fraction did not reproduce this response. WBE and the BPF restored the colonic mucus layer. Particularly, the polymeric PACs-rich F3 fraction increased the mucin-secreting goblet cells number. WBE caused a significant 2-fold higher proportion of Adlercreutzia equolifaciens whereas oligomeric PACs-rich F2 fraction increased by 2.5-fold the proportion of Akkermansia muciniphila. This study reveals the key role of WBE PACs in modulating the gut microbiota and restoring colonic epithelial mucus layer, providing a suitable ecological niche for mucosa-associated symbiotic bacteria, which may be crucial in triggering health effects of blueberry polyphenols.