Nitrogen (N) is an essential element usually limiting in plant growth and a basic factor for increasing the input cost in agriculture. To ensure the food security and environmental sustainability it is urgently required to manage the N fertilizer. The identification or development of genotypes with high nitrogen utilization efficiency (NUE) which can grow efficiently and sustain yield in low N conditions is a possible solution. In this study, two isogenic rice genotypes i.e., wild-type rice kitaake and its transgenic line PP2C9TL overexpressed protein phosphatase gene (PP2C9) were used for comparative proteomics analysis at control and low level of N to identify specific proteins and encoding genes related to high NUE. 2D gel electrophoresis was used to perform the differential proteome analysis. In the leaf proteome, 30 protein spots were differentially expressed between the two isogenic lines under low N level which were involved in the process of energy, photosynthesis, N metabolism, signaling, and defense mechanisms. In addition, we have found that protein phosphatase enhances nitrate reductase activation by downregulation of SnRK1 and 14-3-3 proteins. Furthermore, we showed that PP2C9TL exhibits higher NUE than WT due to higher activity of nitrate reductase. This study provides new insights on the rice proteome which would be useful in the development of new strategies to increase NUE in cereal crops.