Nitrogen (N) is an essential macronutrient for plant growth and development as it is an essential constituent of biomolecules. Its availability directly impacts crop yield. Increased N application in crop fields has caused environmental and health problems, and decreasing nitrogen inputs are in demand to maintain crop production sustainability. Understanding the molecular mechanism of N utilization could play a crucial role in improving the nitrogen use efficiency (NUE) of crop plants. In the present study, the effect of low N supply on plant growth, physio-biochemical, chlorophyll fluorescence attributes, yield components, and gene expression analysis were measured at six developmental stages in rice cultivars. Two rice cultivars were grown with a supply of optimium (120kgha-1) and low N (60kgha-1). Cultivar Vikramarya excelled Aditya at low N supply, and exhibits enhanced plant growth, physiological efficiency, agronomic efficiency, and improved NUE due to higher N uptake and utilization at low N treatment. Moreover, plant biomass, leaf area, and photosynthetic rate were significantly higher in cv. Vikramarya than cv. Aditya at different growth stages, under low N treatment. In addition, enzymatic activities in cultivar Vikramarya were higher than cultivar Aditya under low nitrogen, indicating its greater potential for N metabolism. Gene expression analysis was carried out for the most important nitrogen assimilatory enzymes, such as nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), and glutamate synthase (GOGAT). Expression levels of these genes at different growth stages were significantly higher in cv. Vikramarya compared to cv. Aditya at low N supply. Our findings suggest that improving NUE needs specific revision in N metabolism and physiological assimilation. Overall differences in plant growth, physiological efficiency, biochemical activities, and expression levels of N metabolism genes in N-efficient and N-inefficient rice cultivars need a specific adaptation to N metabolism. Regulatory genes may separately or in conjunction, enhance the NUE. These results provide a platform for selecting crop cultivars for nitrogen utilization efficiency at low N treatment.