To utilize limited nitrogen (N) in terrestrial ecosystems, different plant types may employ different N uptake strategies to avoid and reduce competition for resources and thus complete their life cycles. It is helpful to study whether there is niche separation in the use of N by different plant types in desert ecosystems, for gaining insights into the survival strategies of desert plants and for better understanding the effects of N on their survival. Centaurea pulchella, Lactuca undulata (both ephemeral plants), Ceratocarpus arenarius, and Suaeda glauca (both annual plants) are four widely distributed plant species in the Gurbantunggut Desert in northwest China. We conducted an experimental field study employing the 15N labeling technique to study the uptake strategy of different N forms by these two types of plants in different months and different soil layers. The results indicated that the N uptake rates during the maximum biomass period of the two plant types were higher than those during the fastest growth period in different soil depths. Although all four plants could absorb various common forms of N, inorganic N was still the major form of N absorbed by them. Ephemeral plants preferred to absorb nitrate, with the highest uptake rate reaching 5.74 μg h−1. However, annual plants preferred to absorb ammonium at a maximum uptake rate of 4.74 μg h−1. According to the contribution rate of different N forms to total N uptake, nitrate was the most favorable form for ephemeral plants (contribution rate 36.98%–48.13%), whereas ammonium was the most favorable form for annual plants with the highest and lowest contribution rates of 48.3% and 30.47%, respectively. Furthermore, rather than the plants’ growth characteristics, factors such as month, soil depth, and plant life-form were the key drivers influencing the preferences for different forms of 15N. The research revealed that ephemeral and annual desert plants exhibit diversity in their utilization and recovery of N, and all of them could also absorb the soluble organic N source in the Gurbantunggut Desert. Our study contributed to a better understanding of plasticity of nutrient utilization by different plant species under N constraints in desert ecosystems.