Phytoremediation of arsenic contamination using the hyperaccumulator Pteris vittata is an active area of research. However, the mechanism for the uptake of arsenic in P. vittata remains unclear, due to the absence of methods to monitor this process. This study utilized synchrotron radiation X-ray fluorescence (XRF) spectroscopy and X-ray absorption near-edge structure (XANES) to continuously observe in situ arsenic in living P. vittata. The results showed that the reduction of arsenic(V) to arsenic(III) occurred simultaneously with an increase in frond arsenic, suggesting the reduction affected arsenic uptake. In situ XRF measurements of living P. vittata accurately characterized arsenic uptake but in situ XANES could not be used on live plants without using methods to prevent radiation damage. A novel approach is reported to investigate the changes of elements in plants.