AbstractMicrobial necromass carbon (C) has been considered an important contributor to persistent soil C pool. However, there still lacks large‐scale systematic observations on microbial necromass C in different soil layers, particularly for alpine ecosystems. Besides, it is still unclear whether the relative importance of biotic and abiotic variables such as plant C input and mineral properties in regulating microbial necromass C would change with soil depth. Based on the combination of large‐scale sampling along a ~2200 km transect across Tibetan alpine grasslands and biomarker analysis, together with a global data synthesis across grassland ecosystems, we observed a relatively low proportion of microbial‐derived C in Tibetan alpine grasslands compared to global grasslands (topsoil: 45.4% vs. 58.1%; subsoil: 41.7% vs. 53.7%). We also found that major determinants of microbial necromass C depended on soil depth. In topsoil, both plant C input and mineral protection exerted dominant effects on microbial necromass C. However, in subsoil, the physico‐chemical protection provided by soil clay particles, iron‐aluminum oxides, and exchangeable calcium dominantly facilitated the preservation of microbial necromass C. The differential drivers over microbial necromass C between soil depths should be considered in Earth system models for accurately forecasting soil C dynamics and its potential feedback to global warming.