Human activities have greatly influenced the natural nitrogen cycle, causing dramatic degradation of ecosystem function. Net anthropogenic nitrogen input (NANI) is an important factor contributing to the impact of human activities on the regional nitrogen cycle. Here, we analyzed the temporal and spatial variation of NANI in the Pearl River Basin of China between 1986 to 2015, and found that the total amount of NANI significantly increased from 3,362.25 kg N km -2 yr -1 to 8,071.15 kg N km -2 yr -1 . Application of nitrogen fertilizers was the largest component of NANI in the Basin, accounting for 55.53% in the total NANI, followed by food/feed net nitrogen input (21.26%), atmospheric nitrogen deposition (12.95%), and crop nitrogen fixation (10.26%). Over the last three decades, nitrogen inputs from atmospheric nitrogen deposition have become the second largest source of NANI due to rapid industrialization and urbanization in the region. Regression analysis showed that the rapid growth of both GDP and population density were the main contributors to the increase of NANI. In addition, the increase in the number of red tides in the Pearl River Estuary was strongly correlated with NANI discharge (R 2 = 0.90, p<0.01), suggesting the NANI's eutrophication effect. In total, this study provides a quantitative understanding of the temporal and spatial variations of NANI in the Pearl River Basin as well as the effects of NANI on estuarine waters, and offered key information for developing an integrated strategy for watershed nitrogen management.