Soil acidification is a global environmental problem with significant impacts on agricultural production, environmental protection, and ecosystem health. Soil acidification is widespread in China, affecting crop yields, agricultural product quality, and biodiversity. Since the 1980s, much work has been done on acidic soils in China, but it is controversial whether excessive nitrogen fertilizer application can lead to soil acidification mechanisms. To address the above issues, we conducted a meta-analysis of 115 published papers to integrate and analyze the effects of N fertilizer application on soil acidification and biological properties from 1980 to 2024. We also quantified the effect of nitrogen fertilization on soil acidification and biological changes under different climatic conditions. The results showed that under long-term application of nitrogen fertilizers in China from 1980 to 2024, soil pH decreased by an average of 15.27%, and the activities of soil urease, nitrate reductase, nitrite reductase, catalase, glutamate dehydrogenase, and glutamate synthetase decreased by an average of 9.82-22.37%. The soil microbial community richness (Chao1 index) increased by 6.53%, but the community diversity (Shannon index) decreased by 15.42%. Among the dominant soil microorganisms, the relative abundance of bacteria decreased by an average of 9.67-29.38% and the abundance of gene expression of nifH, amoA-AOA, amoA-AOB, and qnorB decreased by 9.92-19.83%. In addition, we found that the mean annual temperature and rainfall impacted soil acidification via their effect on soil microbial diversity and community composition. This study provides a scientific basis for an in-depth understanding of the spatial and temporal variation of soil acidification and biological properties in China.