Dendrobium officinale is an orchid with medicinal and nutritional properties that has received increasing attention because of its health benefits; however, there is limited information about the metabolic basis of these properties. In this report, secondary metabolites and the antioxidant activity of D. officinale stem samples from three provenances were analyzed, using a UHPLC-QqQ-MS/MS-based metabolomics approach. In total, 411 metabolites were identified including 8 categories such as flavonoids and phenolic acids, 136 of which were differential metabolites. These differentially accumulated metabolites (DAMs) were mainly enriched in secondary metabolic pathways such as flavone, flavonol, tropane, piperidine, pyridine, isoquinoline alkaloid biosynthesis and tyrosine metabolism. The metabolomic profiling suggested that the quantity and content of flavonoid compounds accounted for the highest proportion of total metabolites. Hierarchical cluster analysis (HCA) showed that the marker metabolites of D. officinale from the three provenances were mainly flavonoids, alkaloids and phenolic acids. Correlation analysis identified that 48 differential metabolites showed a significant positive correlation with antioxidant capacity (r ³ 0.8 and p < 0.0092), and flavonoids were the main factors affecting the different antioxidant activities. It is worth noting that quercetin-3-O-sophoroside-7-O-rhamnoside and dihydropinosylvin methyl ether might be the main compounds causing the differences in antioxidant capacity of Yunnan provenance (YN), Zhejiang provenance (ZJ), and Guizhou provenance (GZ). These finding provides valuable information for screening varieties, quality control and product development of D. officinale. Copyright © 2023 Luo, Liu, Nie, Huang, Luo, Sun, Ma, Yu and Du.