Under the dual impact of global climate change and intensifying human activities, atmospheric nitrogen deposition has become a key link in the global nitrogen cycle. The significant effects of nitrogen deposition on ecosystem nitrogen cycling have been confirmed. In particular, the effect of increasing nitrogen deposition on soil nitrogen transformation process has been widely studied. However, the response characteristics and regulatory factors of soil nitrogen mineralization rate to nitrogen deposition at the aggregate level are still unclear presently. Especially, there is a lack of direct evidence in semi-arid grasslands that are sensitive to climate change. In order to make up for this shortcoming, this study aimed to explore the response mechanism and regulatory factors of nitrogen mineralization of soil aggregates with different grain sizes to nitrogen addition. In this study, soil samples from different levels of nitrogen addition were collected from the simulated nitrogen deposition control experimental platform of typical grassland on the Loess Plateau, and three grain size aggregates were screened, including large (>2 mm), medium (0.25—2 mm) and small (<0.25 mm) particle-size aggregates. The physicochemical properties (pH, nutrient content), microbial biomass and extracellular enzyme activity of soil in aggregates of different particle sizes were analyzed, and the nitrogen mineralization rate of aggregates was determined by laboratory culture experiments, and the factors affecting the nitrogen mineralization rate were analyzed. The results showed that with the increase of nitrogen addition concentration, the nitrification rate and net nitrogen mineralization rate of the three particle size aggregates increased, and the ammonification rate first decreased and then increased in the large and small aggregates, and increased first and then decreased in the medium aggregates. Nitrogen addition significantly decreased the pH of soil aggregates, and changed soil nutrient content, microbial biomass, enzyme activity and their stoichiometric ratios, but the influencing characteristics were different among the aggregates of different grain sizes, such as the enzyme activity of β-1,4-N-acetylglucosaminidase decreased first and then increased in small and medium aggregates, and continued to increase in large aggregates. The results of random forest analysis showed that the rate of soil nitrogen mineralization after nitrogen addition was mainly affected by soil biological factors in large aggregates, soil physicochemical factors in small aggregates, and In the middle aggregates, it was mainly influenced by both factors. This study emphasized the aggregate particle size effect of nitrogen addition on the regulation of nitrogen mineralization rate in semi-arid grassland, which provides a reference for sustainable management of grassland on the Loess Plateau under the background of global change.