Low-molecular-weight thiol (LMWT) synthesis has been reported to be directly induced by arsenic (As) in Pteris vittata, an As hyperaccumulator. Sulphur (S) is a critical component of LMWTs. Here, the effect of As treatment on the uptake and distribution of S in P. vittata was investigated. In P. vittata grown under low S conditions, the presence of As in the growth medium enhanced the uptake of SO42−, which was used for LMWT synthesis in fronds. In contrast, As application did not affect SO42− uptake in Nephrolepis exaltata, an As non-hyperaccumulator. Moreover, the isotope microscope system revealed that S absorbed with As accumulated locally in a vacuole-like organelle in epidermal cells, whereas S absorbed alone was distributed uniformly. These results suggest that S is involved in As transport and/or accumulation in P. vittata. X-ray absorption near-edge structure analysis revealed that the major As species in the fronds and roots of P. vittata were inorganic As(III) and As(V), respectively, and that As–LMWT complexes occurred as a minor species. Consequently, in case of As accumulation in P. vittata, S possibly acts as a temporary ligand for As in the form of LMWTs in intercellular and/or intracellular transport (e.g. vacuolar sequestration).