Little information is available on the physiological and molecular adaptations of plant roots to long-term copper (Cu) excess revealed by combined analysis of physiology, widely targeted metabolome and RNA-Seq. 'Shatian' pummelo [Citrus grandis (L.) Osbeck] seedlings were supplied six times weekly with nutrient solution at a Cu concentration of 400 (excessive Cu) or 0.5 (control) mu M for six months. Thereafter, excessive Cu impacts on gene expression, metabolites, cell wall (CW) materials and components, as well as some other physiological param-eters in roots were examined. We detected 350 upregulated and 156 downregulated genes, and 142 [90 sec-ondary metabolites (SMs) and 52 primary metabolites (PMs)] upregulated and 48 (31 SMs and 17 PMs) downregulated metabolites in 400 mu M Cu-treated roots (RCu400). Our results suggested that the physiological and molecular adaptions of roots to Cu excess included: (a) increased biosynthesis and accumulation of SMs (lignin, alkaloids, flavonoids, lignans and coumarins, phenolic acids and total phenolics); (b) elevated Cu retention in root CW and reduced Cu transfer to shoots due to increased thickness and lignification of root CW; (c) downregulated and upregulated expression of Cu uptake-and homeostasis-related genes, respectively; and (d) improved capacity to maintain iron homeostasis in cytoplasm and energy homeostasis due to increased accu-mulation of carbohydrates and organic acids and production of ATP. Although the abundances of many me-tabolites and the expression levels of some genes related to the detoxification of reactive oxygen and nitrogen species were elevated in RCu400, they could not protect these roots from oxidative damage. Also, we observed some similarities and differences in excessive Cu-responsive genes and metabolites between leaves and roots.