The root system plays a crucial role in water and nutrient absorption, making it a significant factor affected by nitrogen (N) availability in the soil. However, the intricate dynamics and distribution patterns of cotton (Gossypium hirsutum L.) root density and N nutrient under varying N supplies in Southern Xinjiang, China, have not been thoroughly understood. A two-year experiment (2021 and 2022) was conducted to determine the effects of five N rates (0, 150, 225, 300, and 450 kg N ha−1) on the root system, shoot growth, N uptake and distribution, and cotton yield. Compared to the N0 treatment (0 kg N ha−1), the application of N fertilizer at a rate of 300 kg N ha−1 resulted in consistent and higher seed cotton yields of 5875 kg ha−1 and 6815 kg ha−1 in 2021 and 2022, respectively. This N fertilization also led to a significant improvement in dry matter weight and N uptake by 32.4% and 53.7%, respectively. Furthermore, applying N fertilizer at a rate of 225 kg N ha−1 significantly increased root length density (RLD), root surface density (RSD), and root volume density (RVD) by 49.6–113.3%, 29.1–95.1%, and 42.2–64.4%, respectively, compared to the treatment without N fertilization (0 kg N ha−1). Notably, the roots in the 0–20 cm soil layers exhibited a stronger response to N fertilization compared to the roots distributed in the 20–40 cm soil layers. The root morphology parameters (RLD, RSD, and RVD) at specific soil depths (0–10 cm in the seedling stage, 10–25 cm in the bud stage, and 20–40 cm in the peak boll stage) were significantly associated with N uptake and seed cotton yield. Optimizing nitrogen fertilizer supply within the range of 225–300 kg N ha−1 can enhance root foraging, thereby promoting the interaction between roots and shoots and ultimately improving cotton production in arid areas.