Long term mining activities can cause significant metal pollution in the environment, thereby showing potential risk to the nearby soil and plants. Elucidating the interfacial processes of trace metals from real contaminated soil to plants including wild plants and rice within the rhizosphere can provide important information on metal biogeochemistry and food safety. The current study aims to explore the spatial distribution and molecular speciation of Cu from soil rhizosphere to plants including indigenous plant species and rice collected from mining-impacted field sites, and reveal the possible uptake mechanisms. X-ray absorption near edge structure (XANES) analysis indicated that Cu was primarily associated with iron oxide and sulfide in soil with a minor proportion of organic complexed species. With plant growth, the Cu-oxalate like organic species in rhizosphere soil significantly increased, with a corresponding decrease in Cu-goethite. Synchrotron-based micro-X-ray fluorescence (μ-XRF) microscopy and X-ray absorption near edge structure (XANES) analysis indicated that most Cu was sequestered around the root surface/epidermis, primarily forming Cu alginate-like species as a Cu-tolerance mechanism for both indigenous plant M. floridulus and rice root. From the root epidermis to the cortex and vascular bundle, more thiol-S bound Cu(I) complex was observed, representing the reduced product of Cu(II) by thiol-S ligand in the root. The absorbed Cu was probably transported from the root to the aerial part as Cu-histidine complex, which was observed in the root xylem. In conclusion, the large retention capacity and reduction of Cu(II) in plant roots alleviated Cu toxicity to plants, which was beneficial for phytostabilizing Cu underground and lowering Cu accumulation in rice grains. These findings showed detailed Cu uptake mechanisms in plants from field contaminated sites, which shed lights on the Cu detoxification process and potential remediation strategies.