The microbial community and soil organic carbon (SOC), which play vital roles in soil fertility and the global C cycle, have been heavily altered due to land‐use changes and long‐term fertilization. However, the effect of long‐term fertilization on the microbial community and SOC in land‐use changed soil is still unclear. In this study, a 26‐year field experiment is conducted to detect the bacterial community and SOC stocks in the soils from meadow grasslands (M), croplands without fertilization (NF), and croplands with fertilization for 13 (F13a) and 26 years (F26a) in the Tibetan Plateau. The results show that land‐use change from meadow grassland to cropland induced a decrease in the SOC stocks of total (TOC), free (FOC) and permanganate‐oxidizable OC (POxC) by 61.8–85.0, 51.1–82.8, and 78.4–95.8%, respectively. Long‐term fertilization increased the SOC stocks by 124.4–419.0%, which was still lower than those in the M soils. In addition, macroaggregates (MAA) and bacterial diversity displayed reductions when the land‐use was changed from grassland to cropland, but they were enhanced after long‐term fertilization. Land‐use change and long‐term fertilization both altered the microbial community. MAA served as a habitat for the microbial community and physical protection for SOC. This may be a key driver of changes in the bacterial community and SOC. This study demonstrates that long‐term fertilization alters the microbial community but fails to restore SOC stocks to the level of uncropped meadow soils. Long‐term fertilization integrated with macroaggregates are required to improve OC sequestration for developing sustainable agriculture and mitigating global climate change.