Arsenate (AsV) and arsenite (AsIII) are two dominant arsenic species in the environment. While arsenate uptake is via phosphate transporter in plants, including arsenic hyperaccumulator Pteris vittata , AsIII uptake mechanisms by P. vittata are unclear. In this study, we investigated AsIII uptake by P. vittata involving root radial transport from external medium to cortical cells and xylem loading. In the root symplastic solution, AsIII was the predominant species (90-94%) and its concentrations were 1.6-21 times those in the medium. AsIII influx into root symplast followed Michaelis-Menten kinetics with K(m) of 77.7 μM at external AsIII concentrations of 2.6-650 μM. In the presence of metabolic inhibitor 2,4-dinitrophenol (DNP), arsenic concentrations in the root symplast were reduced to the levels lower than in the medium, indicating that a transporter-mediated active process was mainly responsible for AsIII influx into P. vittata roots. Unlike radial transport, AsIII loading into xylem involved both high- and low-affinity systems with K(m) of 8.8 μM and 70.4 μM, respectively. As indicated by the effect of 2,4-DNP, passive diffusion became more important in arsenic loading into xylem at higher external AsIII. The unique AsIII uptake system in P. vittata makes it a valuable model to understand the mechanisms of arsenic hyperaccumulation in the plant kingdom.