Pteris vittata L. is known as an arsenic hyper-accumulator. To understand the tolerance mechanism, this study investigated the speciation and distribution of chromium accumulated in fern using synchrotron-radiation X-ray fluorescence analysis. The plants were separately treated with K₂Cr₂O₇ and CrCl₃·H₂O in a hydroponic system. The chemical speciations of chromium and sulfur in the fern were carried out by X-ray absorption near edge structure (XANES) analysis; the elemental distributions in their tissues were visualized by micro-XRF imaging. Chromium was highly accumulated in the roots. μ-XANES and XANES indicated that highly toxic Cr(VI) was reduced Cr(III) as less-toxic Cr(III). The fern-cultivated Cr(III) was accumulated as Cr(III). The chemical speciation of sulfur showed that the ratio of S(-II) compounds, such as glutathione (GSH) and cysteine in the roots, increased when was treated with Cr(VI) and Cr(III). This result demonstrated that sulfur had a certain function in the detoxification process of P. vittata L.