ABSTRACT Soil nitrogen (N) retention plays a crucial role in determining the ecosystem capacity to buffer anthropogenic N inputs and provides a sustainable N supply. However, the effect of acidification, driven by atmospheric deposition of N and sulfur (S), on the retention and fate of allochthonous N across soil aggregate size classes remains poorly understood. We utilized a soil‐acidification gradient induced by 0–50 g S m −2 year −1 addition to investigate 15 N recovery in soil N pools within aggregates 21 days after labeling in a Eurasian meadow. Macroaggregates showed higher 15 N recovery in microbial biomass, amino acids, amino sugars, and therefore total N (TN), as well as greater sensitivity of the former two fractions to acidification compared to microaggregates. This was accompanied by higher N hydrolases and net N mineralization in macroaggregates, supporting the aggregate hierarchical theory. Under moderate acidification (pH decrease from 7 to 6), 15 N retention in hydrolyzable ammonium, amino sugars, non‐hydrolyzable N, and TN decreased, likely due to lower microbial immobilization and entombing of allochthonous N. Conversely, severe acidification (pH decrease below 6) enhanced 15 N retention in these N fractions through stabilization of microbial necromass, revealing a non‐linear relationship between acidification and 15 N retention. Concentrations of autochthonous organic N fractions remained unchanged after five‐year acidification. These findings underscore the mediating role of soil microbes across aggregates in allochthonous 15 N retention among N fractions with contrasting bioavailability under acidification.