Abstract: Nitrogen mineralization is a key process affecting nitrogen availability and nitrogen cycling in soils. The desert steppe is a grassland ecosystem with poor resources, a harsh climate, and a relatively simple vegetation structure. Therefore, it is important to study nitrogen availability on the desert steppe to maintain the stable development of these grasslands. In this study, the net nitrogen mineralization rate was monitored over the whole growing season by using the top-buried tube culture method. We monitored the net nitrogen mineralization rate under four grazing treatments [0 (CK), 0.91 (light grazing), 1.82 (moderate grazing), 2.71 (heavy grazing) sheep/(hm²· a)], and analyzed the relationships between environmental factors (soil temperature and humidity) and soil nitrogen content and mineralization. The aim of these analyses was to provide reference data to develop strategies for the sustainable utilization of nitrogen in the desert grassland ecosystem. The soil net nitrogen mineralization rate first increased and then decreased during the growing season, and the effect of stocking rate was higher in July and August in the peak growth period. The net nitrogen mineralization rate was highest in the moderate grazing treatment [1.82 sheep/(hm²·a)] and lowest in the heavy grazing treatment [2.71 sheep/(hm²· a)]. Pearson’s correlation analyses showed that the nitrification rate was positively correlated with net nitrogen mineralization rate, and the net nitrogen mineralization rate was greatly affected by nitrification during the whole growing season. There was a significant negative correlation between soil water content and net nitrogen mineralization rate, but not between soil temperature and net nitrogen mineralization rate. In summary, the moderate grazing treatment [1.82 sheep/(hm²·a)] represents the ideal stocking rate to maintain a stable nitrogen mineralization rate in desert steppe grasslands.