Understanding the arsenic (As) detoxification mechanisms employed by the newly discovered As hyperaccumulator Chinese Brake fern (Pteris vittata L.) is important to optimize As accumulation capability. The present experiment was carried out to determine the location of As reduction and thiol formation in Chinese Brake fern and the ability of excised plants to absorb As under P influence. Live Chinese Brake ferns were separated into three parts: pinnae (leaflets), fronds (aboveground biomass, de-rooted), and roots (belowground biomass, de-topped). The excised plants were then exposed to 0.5-strength Hoagland nutrient solutions containing 667 M As (III), As (V), or monomethylarsonic acid (MMA) and 0 M P or 500 M P for 1 day. Arsenate and arsenite were separated using an As speciation cartridge and As was determined by graphite furnace atomic absorption spectrophotometry (GFAAS). The pinnae absorbed the greatest amounts of As followed by fronds and roots. In the presence of P, the phytoavailability of As species was As (III)>MMA>As (V) for pinnae and roots and As (V)>As (III) ≌MMA for fronds. The fact that As (III) was the predominant form in excised aerial tissues whereas As (V) was the main form in excised roots clearly demonstrated that As reduction occurred mostly in the fronds, mainly in the pinnae. Absorption of As species resulted in formation of thiol, with MMA causing the greatest level of formation. Although addition of P to the solution suppressed As (V) accumulation in excised pinnae and roots, it enhanced As (V) reduction, and reduced thiol production. The results suggested that the ability to efficiently reduce As (V), facilitated by P, and to quickly produce thiols might have both contributed to the capability of Chinese Brake fern to hyperaccumulate As.