Globally only ≈50% of applied nitrogen (N) fertilizer is captured by crops, and the remainder can cause pollution via runoff and gaseous emissions. Synchronizing soil N supply and crop demand will address this problem, however current soil analysis methods provide little insight into delivery and acquisition of N forms by roots. We used microdialysis, a novel technique for in situ quantification of soil nutrient fluxes, to measure N fluxes in sugarcane cropping soils receiving different fertilizer regimes, and compare these with N uptake capacities of sugarcane roots. We show that in fertilized sugarcane soils, fluxes of inorganic N exceed the uptake capacities of sugarcane roots by several orders of magnitude. Contrary, fluxes of organic N closely matched roots' uptake capacity. These results indicate root uptake capacity constrains plant acquisition of inorganic N. This mismatch between soil N supply and root N uptake capacity is a likely key driver for low N efficiency in the studied crop system. Our results also suggest that (i) the relative contribution of inorganic N for plant nutrition may be overestimated when relying on soil extracts as indicators for root-available N, and (ii) organic N may contribute more to crop N supply than is currently assumed.