Stomatal conductance controls the ability of leaves to acquire carbon sources (CO₂), and it is closely related to crop productivity by affecting photosynthesis. In view of the current situation of slowing N use efficiency caused by N fertilizer overuse in paddy soil, it is necessary to study the regulation strategy of high N fertilizer input to promote N absorption and utilization. Therefore, ZmK2;1 overexpression transgenic lines were used to set up a field experiment of three conditions including N deficient (LN, no N application), moderate or reduced N input (MN, 200 kg/hm²) and N fertilizer overuse (HN, 350 kg/hm²), which were used to study the N nutrition characteristics and production traits in the later growth stage. The results show that ZmK2;1 overexpression can improve the yield characteristics for rice plants under each N application level. At the same time, the content of N and K are increased differentially, it optimizes nitrogen nutrition characteristics of the whole plant and key functional leaves (flag leaves), and promotes the accumulation of plant biomass. Besides, the flag leaf of ZmK2;1 overexpression plant maintains a promising photosynthetic rate (Pn) and stomatal conductance (g_s) during the later growth period. In addition, ZmK2;1 overexpression can promote the N use efficiency of rice under the N fertilizer moderate input and N fertilizer overuse, especially under the condition of N fertilizer overuse, the overexpression of this gene can still promote agronomic N use efficiency (AE_N), physiological N use efficiency (PE_N) and N harvest index (NHI). Obviously, overexpression of the stomatal potassium channel ZmK2;1 in rice can regulate the N/K ratio of plants and key functional leaves, promote photosynthetic rate, and improve the rice yield under different N application. In particular, under the condition of N fertilizer overuse, it can still maintain a high photosynthesis assimilation ability, and plays a positive effect on performing the N use efficiency and yield-increasing potential of rice.