Soil microorganisms are the main drivers in maintaining soil health. This paper focuses on the process of soil mineral ions and microorganisms involved in regulating the phosphorus-sulfur cycle, and systematically investigates the repair and improvement mechanism of soil microorganisms. Relying on an experimental area of a typical grassland in Inner Mongolia, we set up experiments with different nitrogen addition treatments, and combined with one-way analysis of variance (ANOVA) to investigate the distribution of soil phosphorus and sulfur fractions under various scenarios. Then, structural equation modeling was applied to explore the dynamic role between microbial action and phosphorus-sulfur cycle under N addition. Under different nitrogen addition scenarios, Ca10-P accounted for the largest proportion of inorganic phosphorus fractions, which were all greater than 40%. The percentage of inorganic sulfur in the soil was relatively small, less than 3% of total sulfur, and the response of inorganic and total sulfur to the gradient of nitrogen addition, nitrogen frequency, and different grassland management practices was not obvious. Fungal communities were important drivers of changes in functional genes for interleaf phosphorus and sulfur cycling at different N application levels, i.e., N fertilizer application altered the interleaf fungal communities by affecting soil physicochemical properties, which significantly regulated the interleaf bacterial communities, phosphorus and sulfur cycling functional gene abundance, and pathogenic fungal abundance.