Multi-elements hyperaccumulators are of high scientific interest to be applied in remediation of mix-contaminated soils. Sedum alfredii is a well-known Cd/Zn co-hyperaccumulator with high Pb and Cu tolerance. This study investigated the ability of the hyperaccumulating ecotype (HE) S. alfredii to tolerate and accumulate Ni. Differed from the non-hyperaccumulating ecotype (NHE), HE plants grew healthy after 50 µM Ni exposure for 4 weeks. The HE plants translocated up to 40 % Ni to the shoots under high Ni stress and accumulated >3000 and 200 mg kg⁻¹ Ni in roots and shoots, respectively. Micro-XRF image showed that Ni was highly restricted within the HE stem and leaf vascular bundles, especially the xylem tissues. The HE roots were of high Ni tolerance, showing much less pronounced Ni-induced phytotoxicity as compared with the NHEs. Ni-induced O₂– was observed in the apoplastic part of HE root cells, but both Ni and the induced O₂– were highly accumulated in the sensitive zone (root cap, meristem, and cylinder) of NHE roots. These results suggest that although low Ni mobility out of vascular tissues limits the metal accumulation in stems and leaves, HE S. alfredii is highly tolerant towards Ni stress by metal homeostasis in root cells.