Nitrogen is an important nutrient for plant growth and essential metabolic processes. Roots integrally obtain nutrients from soil and are closely related to the growth and development of plants. In this study, the morphological analysis of rice root tissues collected at different time points under low-nitrogen and normal nitrogen conditions demonstrated that, compared with normal nitrogen treatment, the root growth and nitrogen use efficiency (NUE) of rice under low-nitrogen treatment were significantly improved. To better understand the molecular mechanisms of the rice root system’s response to low-nitrogen conditions, a comprehensive transcriptome analysis of rice seedling roots under low-nitrogen and control conditions was conducted in this study. As a result, 3171 differentially expressed genes (DEGs) were identified. Rice seedling roots enhance NUE and promote root development by regulating the genes related to nitrogen absorption and utilization, carbon metabolism, root growth and development, and phytohormones, thereby adapting to low-nitrogen conditions. A total of 25,377 genes were divided into 14 modules using weighted gene co-expression network analysis (WGCNA). Two modules were significantly associated with nitrogen absorption and utilization. A total of 8 core genes and 43 co-expression candidates related to nitrogen absorption and utilization were obtained in these two modules. Further studies on these genes will contribute to the understanding of low-nitrogen adaptation and nitrogen utilization mechanisms in rice.