• The soil aggregate stability increased with increasing duration of vegetation restoration.
• Natural restoration has a positve effect on soil microbial diversity was generally higher in large particle size aggregates, which leads to low environmental stress and strong stability.
• Microorganism continually changed their regulation of pathways as their environment changed.
• Environment adaptability influences soil physiological indicators to varying degrees.
• After years of natural restoration, the soil microbial community generally transformed from nutrient-rich to heterotroph-dominan.
Soil aggregate fractions can regulate microbial community composition and structure after vegetation restoration. However, there has been less focus on the effects of soil aggregate fractions on the distributions of microbial communities. Here, we used phospholipid fatty acid (PLFA) analysis to explore the effects of different years of vegetation restoration (a 35-year-old Thymus mongolicus community (Re-35yrs) and a 2-year-old nongrazing grassland (Ug-2yrs)) on microbial communities within different soil aggregate sizes (<0.25 mm, 0.25–1 mm, 1–2 mm, 2–3 mm, 3–5 mm and>5 mm). The results indicated that the amount of total PLFA in Re-35yrs was 10 times greater than that in Ug-2yrs. The soil aggregate stability increased with increasing duration of vegetation restoration. In Re-35yrs, the total PLFA shown an increase as the soil aggregate size increased, and the highest values were observed in 3~5 mm. Ug-2yrs differed from Re-35yrs, the soil microbial diversity was higher in medium particle sizes (1–2 mm and 2–3 mm) and lower in microaggregates (<0.25 mm and 0.25–1 mm) and macroaggregates (3~5 mm and>5 mm). Soil microbial diversity was highest in large particle size aggregates, which resulted in low environmental stress and strong stability. The same tendency was observed in the high values of cyc/prec, S/M and soil organic matter, which indicated a lower turnover speed (F/B) of fungal energy utilization and a higher fixation rate. After years of natural restoration, the soil microbial community generally transformed from nutrient-rich to heterotroph-dominant, especially in microaggregates (reflected in the G+/G− ratio).