Nitrogen (N) delivered by rivers causes severe eutrophication in many coastal waters, and its turnover and retention are therefore of major interest. We set up a mass balance along a 582 km river section of a large, N-rich lowland river to quantify N retention along this river segment and to identify the underlying processes. Our assessments are based on four Lagrangian sampling campaigns performed between 2011 and 2013. Water quality data served as a basis for calculations of N retention, while chlorophyll-a and zooplankton counts were used to quantify the respective primary and secondary transformations of dissolved inorganic N into biomass. The mass balance revealed an average N retention of 17 mg N m−2 h−1 for both nitrate N (NO3–N) and total N (TN). Stoichiometric estimates of the assimilative N uptake revealed that, although NO3–N retention was associated with high phytoplankton assimilation, only a maximum of 53% of NO3–N retention could be attributed to net algal assimilation. The high TN retention rates in turn were most probably caused by a combination of seston deposition and denitrification. The studied river segment acts as a TN sink by retaining almost 30% of the TN inputs, which shows that large rivers can contribute considerably to N retention during downstream transport.