Abstract: 【Objective】 To analyze changes of metabolites of Anoectochilus roxburghii(Wall.) Lindl. after lead stress, to study response mechanism of A. roxburghii to lead stress.【Method】 After lead stress treatment, A. roxburghii seedlings were sampled at different time(0, 7, 14 and 30 d) to determine content of active substances such as flavonoids, soluble sugars, free amino acids, aureoside, and analyze effects of lead stress on A. roxburghii metabolites.【Result】 As lead stress went on, contents of 2 flavones(rutin and narcissin) and soluble sugar first increased and then decreased. 21 free amino acids were detected in A. roxburghii, and aspartic acid content was the highest;proline(Pro) content increased rapidly after stress and remained high;and components of other free amino acids started to increase significantly until 14 d (P<0.05). The results of non-targeted metabolomics showed that on the 7, 14 and 30 d, numbers of differential metabolites were 92, 108 and 67, respectively, of which 6, 32 and 10 were up-regulated and 86, 76, 57 were down-regulated. Differential metabolites were mainly fatty acyl, amino acids and their derivatives, prenol phospholipids and organic acids. Metabolic pathways with highly significant differences included N-glycan biosynthesis(up-regulated), carotenoid biosynthesis(up-regulated), brassinosteroid biosynthesis, valine, leucine and isoleucine degradation, and biosynthesis of saturated fatty acids.【Conclusion】 In the early lead stress, A. roxburghii can reduce stress damage by regulating lipid metabolism, amino acid metabolism and synthesis of flavonoid compounds, but long-term high-content lead stress will lead to the decrease of content of main component such as flavone, amino acid and kinsenoside.