BACKGROUND: Dark tea, comprising one of the six major teas, has many biological activities, which originate from their active substrates, such as polyphenols, polysaccharides, and so on. The hypoglycemic effect is one of its most prominent activities, although less is known about their evaluation and potential role in the hypoglycemic mechanism.RESULTS: In the present study, we separately analyzed the phytochemical composition, glycosidase inhibition and free radical scavenging activities, and hypoglycemic activity in type 2 diabetes mellitus mice, as well as the alleviation of insulin resistance in HepG2 cells of four dark tea aqueous extracts. The results showed that the phytochemical composition of dark tea aqueous extracts was significantly different, and they all had good glycosidase inhibition and free radical scavenging activities, in vivo hypoglycemic activity and alleviation of insulin resistance, and could also activate the phosphatidylinositol 3-kinase-Aktperixisome proliferation-activated receptor cascade signaling pathway to regulate glucose and lipid metabolism, change the key enzyme activities related to glucose metabolism and antioxidant activity, and reduce oxidative stress and inflammatory factor levels. Among them, Liubao brick tea (LBT) and Pu-erh tea (PET) possessed better glycosidase inhibitory activity, in vivo hypoglycemic activity and improved insulin resistance activity, whereas Qingzhuan brick tea and Fuzhuan brick tea had better free radical scavenging activity, which may be explained by their distinct phytochemical compositions, such as tea proteins, polysaccharides, polyphenols, catechins, and tea pigments and some elements.CONCLUSION: Dark tea is a highly attractive candidate for developing antidiabetic food, LBT and PET may be good natural sources of agricultural products with anti-diabetic effects.