Metal-based nanoparticles (MNPs) may be translocated and biochemically modified in vivo, which may influence the fate of MNPs in the environment. Here, synchrotron-based techniques were used to investigate the behavior of CuO NPs in rice plants exposed to 100 mg/L CuO NPs for 14 days. Micro X-ray fluorescence (μ-XRF) and micro X-ray absorption near edge structure (μ-XANES) analysis revealed that CuO NPs moved into the root epidermis, exodermis, and cortex, and they ultimately reached the endodermis but could not easily pass the Casparian strip; however, the formation of lateral roots provided a potential pathway for MNPs to enter the stele. Moreover, bulk-XANES data showed that CuO NPs were transported from the roots to the leaves, and that Cu (II) combined with cysteine, citrate, and phosphate ligands and was even reduced to Cu (I). CuO NPs and Cu-citrate were observed in the root cells using soft X-ray scanning transmission microscopy (STXM).