Nitrogen and brassinosteroids (BRs) play a vital role in modulating the growth, development, and yield of rice. However, the influences of BRs on nitrogen assimilation and metabolism in rice are not fully understood. In this study, we analyzed the impact of BRs on nitrogen utilization in rice using the indica variety ‘Zhongjiazao 17’ and the japonica variety ‘Nipponbare’ in hydroponic conditions. The results showed that BR treatment could efficiently elevate nitrate and ammonium nitrogen accumulation in both shoots and roots. Furthermore, some genes involved in the uptake of nitrate and ammonium in roots were stimulated by BRs, though we noted subtle variances between the two rice cultivars. Moreover, BRs augmented the activity of nitrate reductase (NR) and glutamine synthetase (GS) in roots, along with NR in shoots. Interestingly, BRs also spiked the total free amino acid content in both the shoots and roots. Gene expression analysis uncovered a robust induction by BRs of NR genes and GS-related genes in the roots of both ‘Nipponbare’ and ‘Zhongjiazao 17’. Collectively, our data suggest that BRs significantly enhance the accumulation of both nitrate and ammonium in rice and trigger a series of reactions related to nitrogen utilization.