Morus nigra L. has been widely used in Brazilian folk medicine for the treatment of diabetes. We evaluate the chemical composition and antidiabetic properties of the hexane (Hex-Mn) and chloroform (Chlo-Mn) fractions obtained by partition of the crude ethanolic extract from the leaves in rats. Chemical composition analysis of Hex-Mn and Chlor-Mn was performed by gas chromatography-mass spectrometry (CG-MS). In vivo and in vitro studies were carried out to compare the antidiabetic activities of the Hex-Mn and Chlor-Mn fractions. Most of the compounds identified in Hex-Mn were α-linolenic acid, stigmast-5-en-3-ol and linolenic acid ethyl ester, while in Chlor-Mn, stigmast-5-en-3-ol, palmitic acid and α-linolenic acid were mainly identified. Only Hex-Mn treatment reduced both fasting and postprandial hyperglycemia. Additionally, Hex-Mn preserved body weight gain, preserved the hepatic glycogen content, and also reduced the thiobarbituric acid reactive substances and nitrite levels, as well as restored the superoxide dismutase. Furthermore, digestion of complex carbohydrates and intestinal glucose absorption was prevented by Hex-Mn treatment. Our results suggest that the antidiabetic activity of Hex-Mn may be explained, at least in part, by the insulin sensitivity increase, antioxidant properties and reduction in carbohydrate absorption in the small intestine.