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Root water uptake is strongly influenced by the morphology and anatomical structure of roots, which are regulated by nitrogen forms and environmental stimuli. To further illustrate the roles of different nitrogen forms on root water uptake under osmotic stress, a split-root system was supplied with different nitrogen forms and osmotic stress simulated by adding 10% (w/v) polyethylene glycol (PEG, 6000). The local effects of nitrogen form and osmotic stress on root morphology, anatomical structure, root lignin content, and water uptake rate were investigated. Under osmotic stress conditions, ammonium markedly promoted the formation and elongation of the lateral root, whereas a significant decrease in numbers of lateral roots was observed under local nitrate supply. Under nitrate supply in split-root systems, osmotic stress significantly promoted root cell death and more aerenchyma formation, as well as accelerated the lignification of the root. However, osmotic stress had no negative effect on the root anatomical structure under ammonium supply. The root water uptake rate was significantly higher in split-root supplied with ammonium than nitrate under osmotic stress conditions. In conclusion, the high water uptake ability in local ammonium supply was associated with the more lateral roots development and the lower cell death, aerenchyma formation and lignification under osmotic stress. • Osmotic stress promoted lateral root development with local ammonium supplied. • Osmotic stress significantly induced aerenchyma formation in local nitrate supply. • Exceeded aerenchyma formation obviously restrict water uptake. • Ammonium could maintain a higher water uptake and increase drought resistance. [ABSTRACT FROM AUTHOR]