In Mexico, Cactaceae plants are widely used in folk medicine for the treatment of diabetes. The genus Opuntia spp. Opuntia matudae Sheinvar prickly pears are known as xoconostle and are used in Mexican cuisine for their acidic flavor. Currently there are few reports of pharmacological properties of this plant, which include antioxidant and antimicrobial activities. This study focuses on the chemical characterization of the methanolic (OmMe) and aqueous (OmAq) extracts and the evaluation of the antidiabetic activity of O. matudae fruits in two biological models. For the in vivo model, streptozotocin (STZ)-induced diabetic mice were used, and for the in vitro model, liver sections isolated from healthy mice were used. The OmAq (100 mg/kg, oral pathway [p.o.]) extract decreased postprandial glucose peak at 0.5 h after glucose uptake by 43.1%, similarly, OmMe (100 mg/kg, p.o.) extract reduced postprandial glucose peak at 0.5 h by 34.1% in healthy mice. The effect of the two extracts and the fraction of the mixture of unidentified betalains (OmB) of O. matudae evaluated in the isolated mouse liver slice model showed a concentration-dependent decrease in hepatic glucose output (HGO) with and without insulin administration with the OmMe extract. The OmAq extract, however, showed concentration-dependent increases of HGO with and without insulin, and the OmB fraction generally exhibited an insulin mimetic effect. Moreover, both OmAq and OmMe extracts were tested in mice with STZ-induced diabetes (160 mg/kg, intraperitoneal route), using a semichronic daily administration (2-28 days after diabetes onset) of OmAq extract was able to reduce blood glucose by 34.3%, meanwhile OmMe extract reduced blood glucose by 22.9%, 28 days after diabetes onset. We identified five compounds (1-5) in the two extracts, consisting of two phenolic acids (1, 2), three flavanols (3-5), as well as two unidentified betalains. Therefore, we conclude that the aqueous extract of the xoconostle fruit where betalains are present may be useful for the treatment of diabetes mellitus.