In China, many estuaries suffer from eutrophication problems such as green tides and hypoxia. This is often a result of human activities on land leading to increased nutrient exports by rivers. River pollution shows seasonal trends that are not well understood. Therefore, the main objective of this study is to improve our understanding of the seasonal variation in river export of dissolved inorganic nitrogen (DIN) by source and at the sub-basin scale. To this end, we modified the existing MARINA model 1.0 (Model to Assess River Input of Nutrient to seAs) to account for seasonality in river export of DIN, and applied it to the Yangtze River. The resulting MARINA model version 1.1 takes a mass-balance approach and accounts for seasonality in human activities (e.g., crop planting and fertilization) and meteorology. The model distinguishes four seasons: winter (December–February), spring (March–May), summer (June–August) and fall (September–November). Our results for Yangtze indicate that N inputs to land and river export of DIN to sea are higher in summer and lower in winter. In spring, summer and fall, diffuse sources from agriculture contribute 43–85% to DIN export. In spring and fall, use of synthetic N fertilizers in cropland is an important source of DIN. In summer, both atmospheric N deposition and synthetic N fertilizers dominate. Animal manure is typically applied on land in spring and fall, contributing then to DIN. In winter, point sources of animal manure are responsible for 34–74% of DIN river export. In general, more DIN is exported to the sea from activities in middlestream and downstream sub-basins. Our results can serve as an example for other large rivers worldwide, and support the formulation of effective strategies to reduce seasonal eutrophication.