Arsenic (As) hyperaccumulation trait has been described in a limited number of fern species. The physiological basis of hyperaccumulation remains unclear, especially in non-Pteris species such as Pityrogramma calomelanos. Aiming at a better understanding of As-induced responses, P. calomelanos plants were exposed to 1 mM As for 21 days and compared with control plants. Chemical analyses revealed that As accumulation was ten times higher in pinnae then in roots and stipes. In pinnae, As was present mainly as arsenite, whereas arsenate was the dominant form in stipes and roots. Arsenic promoted an increase in antioxidant enzyme activities in both fern parts and several alterations in mineral nutrition, especially with regard to P and K. A higher content of non-protein thiols was observed in pinnae of plants exposed to As, whereas As induced the increase in lipid peroxidation in roots. The results showed that Pityrogramma calomelanos shares with Pteris vittata several aspects of As metabolism. High root-shoot As translocation showed to be essential to avoid toxic effects in roots, since the root is more sensitive to the metalloid. The higher capacity of P. calomelanos to sequester arsenite in the pinna and its efficient antioxidant system maintain the reactive oxygen species at a low level, thus enhancing the continuous accumulation of As. Molecular investigations are needed to elucidate the evolution of As-tolerance mechanisms in Pteridaceae species, especially with regard to membrane transporters and ROS signaling.