Modeling studies of nitrogen flux in large rivers, including the Mississippi River, suggest that much of the nitrogen that enters rivers is conserved and exported.  However, patterns of nitrogen cycling, including denitrification, in the Mississippi River are known to be complex and vary with habitat type and season.  In this study, spatial habitat data and empirically derived denitrification rates were used to extrapolate nitrogen loss to the 2,400 km reach of the Mississippi River from Minneapolis, Minnesota to the Atchafalaya River diversion in Louisiana.  Our results indicate a system-wide in-stream denitrification nitrogen loss of 159,044 Mg N y^-1 (about 9.5% of the nitrogen load).  The results also indicated that nitrogen removal was greater in the northern reaches compared to that in the southern reaches because of increased habitat complexity in the northern navigation pools.  The southern reaches contain primarily main channel and side channel habitats; whereas in the northern reaches, impoundments and backwater lakes contribute a combined 40% of the surface area.  Overall, our results are consistent with high throughput of nitrogen, but specify that habitat diversity and river channel complexity are driving factors that influence retention time, depth, and other physical/chemical variables that lead to increased riverine nitrogen loss.