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Liupao tea, a popular dark tea, exhibited anti-diabetic activity in Chinese tea culture. However, the bioavailability of the main active components is low. Therefore, the relationship between the anti-diabetic activity and the modulation of gut microbiota was investigated in this study for the first time. Liupao tea extract (LTE) could significantly alleviate hyperglycemia, insulin-resistance, and plasma lipid profiles. The hypoglycemic effect of high-dose LTE was comparable to that of metformin. Importantly, LTE exhibited beneficial effect on modulating the diabetic-induced disorder of gut microbiota, such as increasing Bacteroidetes/Firmicutes ratio and up-regulating putative short-chain fatty acid (SCFA)-producing bacteria, including Prevotella and Bacteroides. Moreover, 29 key phylotypes were significantly correlated with at least one diabetic index. In conclusion, LTE could be developed into an anti-diabetic functional beverage and its beneficial effects is likely to be associated with the prebiotic effects for increasing the level of SCFAs and the relative abundance of SCFA-producing bacteria.