Abstract: Net anthropogenic nitrogen input (NANI) is one of the critical reasons for the eutrophication of water bodies. Payments for ecosystem services (PES) reduce NANI by changing the production and living behaviors of residents in the basin area to solve the problem of eutrophication of water bodies. The Xin'an River Basin was taken as an example to evaluate the temporal and spatial changes in NANI during the implementation of PES from 2008 to 2017 by calculating N fertilizer application, atmospheric N deposition, agricultural N fixation, and net N food and feed imports. The response of NANI to PES was then examined and it was found that, during the research period, NANI to the Xin'an River Basin showed an overall downward trend. From the perspective of Huangshan city, the value of NANI first rose and then fell, and a significant decline occurred after 2012, that is the period when PES began to be implemented in the Xin'an River Basin. The use of N fertilizer was the main input source of N, and the N content of crop products was the largest item of N output. NANI dropped in Tunxi District and Shexian County. The inflection points of the change occurred were the second stage of PES in Xin'an River and the first stage of PES in Xin'an River, respectively. It could be concluded that PES in the Xin'an River Basin has effectively reduced NANI.