Plants need to be efficient in nutrient management, especially when they face the temporal nutrient defficiencies. Understanding how crops respond to nitrogen (N) starvation would help in the selection of crop cultivars more tolerant to N deficiency. In the present work, the physiological responses of two wheat cultivars, Yannong 19 (YN) and Qinmai 11 (QM), to N starvation conditions were investigated. The two cultivars differed in biomass and N rearrangement between shoots and roots during N starvation. QM allocated more N to roots and exhibited higher root/shoot biomass ratio than YN. However, tissue measurement indicated that both cultivars had similar nitrate content in leaves and roots and similar remobilization rate in roots. Microelectrode measurement showed that vacuolar nitrate activity (concentration) in roots of QM was lower than that in roots of YN, especially in epidermal cells. Nitrate remobilization rates from root vacuoles of two cultivars were also identical. Moreover, vacuolar nitrate remobilization rate was proportional to vacuolar nitrate activity. During N starvation, nitrate reductase activity (NRA) was decreased but there were no significant differences between the two cultivars. Nitrate efflux from roots reduced after external N removal and QM seemed to have higher nitrate efflux rate. Understanding how crops respond to nitrogen (N) starvation would help in the selection of crop cultivars more tolerant to N deficiency. In the present work, the physiological responses of two wheat cultivars, Yannong 19 (YN) and Qinmai 11 (QM), to N starvation conditions were investigated. The two cultivars differed in biomass and N rearrangement between shoots and roots during N starvation. However, tissue measurement indicated that both both cultivars had similar nitrate content in leaves and roots and similar remobilization rates in roots. Microelectrode measurement showed that vacuolar nitrate activity (concentration) in roots of QM was lower than that in roots of YN, especially in epidermal cells. Nitrate remobilization rates from root vacuoles of two cultivars were also identical. Moreover, During nitrate starvation, nitrate reductase activity (NRA) was decreased but there were no significant differences between the two cultivars. Nitrate efflux from roots reduced after external N removal and QM seemed to have higher nitrate efflux rate.