Timescale problems of nitrate behavior (i.e. seasonal variation and long-term trend) in headwater are closely related to its hydrological process. In a typical agricultural headwater catchment in the Chiba prefecture, Japan, the groundwater nitrate concentration showed an increasing trend, while for spring water, a substantial decreasing trend was observed during our monitoring period. Two key issues, (1) identification of multiple flow pathways and (2) evaluation of the residence time for different pathways, were emphasized to reveal the factors controlling the different patterns of nitrate trend. Three major flow pathways including vertical soil water flow (VF), lateral groundwater flow (LG) and deep groundwater flow (DG) along the upland-slope-valley were differentiated. Different timescales of three flow pathways were identified. The residence time of VF was calculated as 9–10 years based on the soil water budget equation and the apparent age of LG was estimated as 41 years by chlorofluorocarbon (CFC) traces. The increasing trend of NO3− in groundwater agreed well with the historical nitrate loading, and the decreasing trend of NO3− in spring was mainly influenced by nitrate behavior of LF, which substantially decreased due to reduction of nitrogen fertilizer loadings since 2000.