ETHNOPHARMACOLOGICAL RELEVANCE: As a traditional health beverage in China, Dark Tea (DT) have been proved to effectively mitigate vascular lesions induced by hyperlipidemia. However, key active ingredient of DT and the potential pharmacological mechanism protecting vascular endothelium is still unclear. AIM OF THE STUDY: This study aimed to investigate the key active ingredient in DT and reveal underlying mechanism responsible for its protective effect on vascular endothelium. MATERIALS AND METHODS: The protective effect of DT on vascular endothelium was evaluated using a high-fat diet-induced zebrafish model. The chemical ingredients of DT were analyzed by ultra-high performance liquid chromatography-quadrupole/time-of-flight mass spectrometry (UHPLC-Q/TOF-MS), and the active ingredients were identified using a multidimensional molecular data mining approach. Molecular biology experiments were used to explore the underlying mechanisms of DT and its active components. RESULTS: The results showed that DT could significantly prevent the deposition of circulatory lipids on the vascular wall, inhibit inflammatory cell aggregation, and reduce microvascular hyperplasia in zebrafish models. An integrated multi-dimensional data mining technique was successfully employed to identify a key active lignan in DT, matairesinol. Furthermore, DT and matairesinol significantly protected endothelial cells by activating AMPK phosphorylation, thereby inhibiting downstream HMGCR protein expression and promoting PPARγ phosphorylation. CONCLUSIONS: Matairesinol has been characterized as a key active ingredient in DT. It protects against high-fat-induced vascular endothelial damage by activating AMPK and downstream signaling pathways. These findings offer new insights into the therapeutic potential of DT as a daily dietary supplement for maintaining vascular health.