In this study, the role of sulfur (S) and arsenic (As) species in the As hyperaccumulator Pteris vittata (P. vittata) and the As hypertolerant plant Adiantum capillus-veneris (A. capillus-veneris) were investigated. Under As exposure, sulfhydryl groups (-SH) increased in both plants, indicating that arsenate enhanced sulfur assimilation. This S then played different roles in the two plants. In A. capillus-veneris, As was mainly coordinated with S; whereas in P. vittata, it was coordinated with oxygen. Differences in As concentration and in the rate of As reduction were noted in the two plants. In A. capillus-veneris, As was present at lower levels and was reduced and coordinated (combined) with -SH. This was considered to represent a defense strategy to limit As transport to the frond. For P. vittata, we deduced that the SH group was likely used as an electron donor to reduce As (V) to As (III), therefore fewer SH groups were used to coordinate with reduced As (III). This was regarded as an accumulation strategy to facilitate As transport. The results of this study suggested that S played important roles in As detoxification and accumulation in tolerant and hyperaccumulating plants.