Cotton is an important global cash crop whose yield and quality are highly influenced by soil nitrogen. Therefore, examining the interactions between roots and arbuscular mycorrhizal fungi (AMF) under reduced nitrogen conditions is of great significance. We investigated the effects of nitrogen application (0, 250, and 375 kg*hm-2) on the AMF infection rate of cotton, the nitrogen content of each organ, root morphological characteristics and biomass, soil extracellular enzyme activity, and soil carbon and nitrogen content using a compartmentalized culture system. The contribution of AMF to plant nitrogen was 10.40, 22.72, and 16.67% under high, low, and no nitrogen treatments, respectively. Under low-nitrogen conditions, the symbiosis between AMF and roots increased root surface area, tip number, branch number, mean diameter, and biomass; and increased soil extracellular enzyme activity (protease, NAG, PER, and PPO), the microbial biomass carbon-to-nitrogen ratio, active carbon content, and the soil nitrogen mineralization rate. Soil NO3--N, NH4+-N, and organic nitrogen content decreased, whereas the absorption of NO3--N by AMF hyphae was higher than that of NH4+-N. Under low-nitrogen conditions, AMF promoted the decomposition of soil organic matter and the transformation of soil nitrogen through the action of hyphal microorganisms.