Field experiments were conducted to study the effects of different drip irrigation lateral layout on the root growth of machine-harvested cotton, water and nitrogen transport, and nitrogen use efficiency. Three drip irrigation capillary configuration methods were set up:(1) Embedded drip irrigation lateral + center layout (EB), (2) embedded drip irrigation lateral + side layout (ES), (3) labyrinth drip irrigation lateral + side layout (LS). The amount of nitrogen (N) applied was 300 kg/hm². At the same time, the ES treatment without nitrogen fertilizer was used as the control (CK). The results showed that after 24 hours of drip irrigation, soil moisture and nitrate nitrogen were mainly distributed in the 0-40 cm deep soil layer. The contents of water and nitrate nitrogen in the root zone below the crop row were high in LS and EB treatments, while the nitrate nitrogen in ES treatment moved to wide rows. More than 90% of cotton roots were distributed in the 0-30 cm soil layer, but the roots of the EB treatment were shallower, and more than 80% of the roots were distributed in the soil layer within 15 cm. Compared with the LS and EB treatments, the dry root matter quality of the ES treatment was significantly reduced by 31.7% and 25.5% respectively. The root length density, root surface area, root volume of ES treatment were significantly higher than those of LS and EB treatments. The LS treatment significantly increased the yield and nitrogen use efficiency, which were 9.4% and 18.0% higher than those of the ES treatment. The yield and nitrogen use efficiency of the EB treatment also increased by 6.5% and 8.5% respectively compared with the ES treatment. Machine-harvested cotton should be planted with the labyrinth drip irrigation lateral and side layout. Then the distribution of water and nitrate nitrogen would match the root system distribution better, which could significantly promote cotton root growth, increase nitrogen uptake, and increase yield and water and nitrogen use efficiency.