Large tropical rivers act as important pathways of nitrogen transport from land to the sea, with the final river reaches potentially retaining nitrogen. In the present study, a mass balance approach was used to evaluate nitrogen retention over a two-year period from a 50 km reach of the Padma River in Bangladesh, approximately 150 km upstream of where the river discharges into the Bay of Bengal. The relationship between concentration and discharge was estimated from 58 nitrogen concentration and discharge measurements. Daily nitrogen flux was then calculated daily from the hydrological inflow and outflows of the reach, and total nitrogen (TN) retention was estimated based on the flux difference of TN inflows and outflows. To compare with mass balance estimates, nitrogen loss due to water retention (NLWR), sedimentation, potential denitrification (PDR), and nitrogen fixation (NFR) were estimated from the water column of the river. NLWR and sedimentation were estimated from the nitrogen concentration and discharge. PDR and NFR were measured using the acetylene inhibition method and upscaled using outflow discharge and water travel time. Up-scaled PDR and NFR were compared with hydrologic retention and sedimentation to assess the relative contributions of these processes to retention. Monthly mass-balance measurements showed a substantial seasonal variation in nitrogen retention. Estimated maximum retention values (tonnes per month) of NLWR, sedimentation, PDR, and NFR were all associated with the monsoons. However, the percentage contribution of PDR and NFR to TN retention was higher in non-monsoon months (post-monsoon, dry/winter and pre-monsoon), suggesting nitrogen retention mechanisms varied seasonally. TN retention via NLWR accounted for the largest portion of total TN retention in every season, always exceeding 50% of total TN retention. Sedimentation was second most important in monsoon and post-monsoon seasons, while PDR in submerged geomorphic units was second most important in dry/winter and pre-monsoon seasons. Net PDR in the water column contributed the least (1-6%) to total TN retention in all seasons.