| Summary: | <i>Morus alba</i> L. is used in traditional Chinese medicine for its anti-diabetic activity; however, the part of the hypoglycemic activity and related active metabolites are still not fully clarified. In this study, the metabolites in the <i>M. alba</i> roots, leaves, twigs, and fruits extracts (70% ethanol extracts) were systematically identified, and their hypoglycemic activity was evaluated by the high-fat diet/streptozotocin-induced 2 diabetes mellitus (T2D) mouse model. A total of 60 high-level compounds, including 16 polyphenols, 43 flavonoids, and one quinic acid, were identified by high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS) combined with the fragmentation pathways of standards and the self-established database. Among them, 23 metabolites were reported for the first time from this plant. In contrast to the extracts of <i>M. alba</i> leaves and fruits, the extracts of roots and twigs displayed significant hypoglycemic activity The glycemia was significantly reduced from 32.08 ± 1.27 to 20.88 ± 1.82 mmol/L and from 33.32 ± 1.98 to 24.74 ± 1.02 mmol/L, respectively, after 4 weeks of treatment with roots and twigs extracts. Compound <b>46</b> (morusin), which is a high-level component identified from the extracts of <i>M. alba</i> roots, also displayed significant activity in decreasing the blood glucose level of T2D mice reduced from 31.45 ± 1.23 to 23.45 ± 2.13 mmol/L. In addition, the extracts of roots and twigs displayed significant activity in reducing postprandial glycemia. This work marks the first comparison of the metabolites and hypoglycemic activity of <i>M. alba</i> roots, leaves, twigs, and fruits extracts, and provides a foundation for further development of <i>M. alba</i> extracts as anti-diabetic drugs.
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