Alyssum corsicum Duby is a candidate plant used for phytomining and phytoremediation studies with its high Ni-accumulating ability of the aboveground tissues. Metal localization and concentration by hyperaccumulator plants and the physiological basis of these phenomena have been of great interest in recent years. A. corsicum seeds were grown at different concentrations of Ni in Murashige and Skoog medium. Multielement concentrations of the plant roots and shoots were determined by polarized energy dispersive X-ray fluorescence. Ni, Ca, Mg, and Fe localization and concentration at the upper and lower leaf surfaces were determined by scanning electron microscopy coupled with the energy-dispersive X-ray analysis technique (SEM-EDX). Results showed that Ni is accumulated more by the shoots than the roots of the plant and that Ni is concentrated mainly in the trichome and the stoma guard cells of the leaves. Trichome density on the leaves was reduced, and some morphological changes of the trichome structure were also observed with increasing Ni levels. Analysis of antioxidant enzyme activities in roots and shoots was performed. The total leaf proteins of the plant were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and differentially expressed genes were detected by specifically designed primers.