Worldwide urbanization has brought dramatic changes in agricultural structures, as well as serious agricultural non-point source pollutions of nitrogen and phosphorus. However, understanding the effect of agricultural structure adjustment on net anthropogenic nitrogen inputs (NANI) has been still limited. In this paper, statistical data from the agricultural statistical Yearbook, the National Economic and Social Development Statistical Bulletin were collected from 1990 to 2019 in the Pearl River Basin, China, and used to analyze the spatial and temporal patterns of NANI and its influencing factors. The results indicated that the agricultural structure adjustment has significantly influenced the spatial and temporal patterns of NANI in the last 30 years in the Pearl River Basin. The NANI decreased from 1990 to 2019, and had a spatial pattern of higher values in the upstream areas and lower in the downstream areas. In terms of the nitrogen input sources of NANI, in the economically developed regions downstream, nitrogen inputs are dominated by food/feed nitrogen, which accounted for an average of 49.6% of total nitrogen inputs. In upstream areas with relatively low economic development, fertilizer nitrogen accounted for an average of 54.9% of total nitrogen inputs. A novel nitrogen input source index of NANI, namely the ratio of agricultural nitrogen inputs to non-agricultural nitrogen inputs of NANI(ASNA), was also proposed to characterize the impact of the agricultural industry restructuring on NANI changes over time. Similar to the characteristics of NANI from 1990 to 2019, the ASNA showed a decreasing trend in the study area. Moreover, agricultural variables (agricultural land area, nitrogen fertilizer consumption and livestock farming density) tended to contribute less to the explained ASNA variances, while the contributions of the non-agricultural factors (population density and non-agricultural GDP) increased from 1990 to 2019. This indicated that the contribution of nitrogen inputs from agricultural sources to the NANI decreased while the contribution of nitrogen inputs from non-agricultural sources increased, with the shifts of agricultural sectors to the secondary and tertiary sectors in the Pearl River Basin. Our findings also suggest that differently regional targeting should be considered for the nitrogen pollution management in the Pearl River Basin, which focuses on the nitrogen pollution management of non-agricultural sources in the downstream areas, and but highlights agricultural nitrogen pollution management in the upstream areas.