Subsoil contains more than half of soil organic carbon (SOC) globally and is conventionally assumed to be relatively unresponsive to warming compared to the topsoil. Here, we show substantial changes in carbon allocation and dynamics of the subsoil but not topsoil in the Qinghai-Tibetan alpine grasslands over 5 years of warming. Specifically, warming enhanced the accumulation of newly synthesized ( 14 C-enriched) carbon in the subsoil slow-cycling pool (silt-clay fraction) but promoted the decomposition of plant-derived lignin in the fastcycling pool (macroaggregates). These changes mirrored an accumulation of lipids and sugars at the expense of lignin in the warmed bulk subsoil, likely associated with shortened soil freezing period and a deepening root system. As warming is accompanied by deepening roots in a wide range of ecosystems, root-driven accrual of slow-cycling pool may represent an important and overlooked mechanism for a potential long-term carbon sink at depth. Moreover, given the contrasting sensitivity of SOC dynamics at varied depths, warming studies focusing only on surface soils may vastly misrepresent shifts in ecosystem carbon storage under climate change.

Subsoils contain >50% of soil organic carbon (SOC) globally yet remain under‐investigated in terms of their response to climate changes. Recent evidence suggests that warmer, drier conditions in alpine grasslands induce divergent responses in SOC decomposition and carbon accrual in top‐ versus subsoils. However, longer term effects on microbial activity (i.e., catabolic respiration vs. anabolic growth) and belowground carbon cycling are not well understood. Here we utilized a field manipulation experiment on the Qinghai‐Tibetan Plateau and conducted a 110‐day soil incubation with and without 13C‐labeled grass litter to assess microbes' role as both SOC “decomposers” and “contributors” in the top‐ (0–10 cm) versus subsoils (30−40 cm) after 5 years of warming and drought treatments. Microbial mineralization of both SOC and added litter was examined in tandem with potential extracellular enzyme activities, while microbial biomass synthesis and necromass accumulation were analyzed using phospholipid fatty acids and amino sugars coupled with 13C analysis, respectively. We found that warming and, to a lesser extent, drought decreased the ratio of inorganic nitrogen (N) to water‐extractable organic carbon in the subsoil, intensifying N limitation at depth. Both SOC and litter mineralization were reduced in the subsoil, which may also be related to N limitation, as evidenced by lower hydrolase activity (especially leucine aminopeptidase) and reduced microbial efficiency (lower biomass synthesis and necromass accumulation relative to respiration). However, none of these effects were observed in the topsoil, suggesting that soil microbes became inactive and inefficient in subsoil but not topsoil environments. Given increasing belowground productivity in this alpine grassland under warming, both elevated root deposits and diminished microbial activity may contribute to new carbon accrual in the subsoil. However, the sustainability of plant growth and persistence of subsoil SOC pools deserve further investigation in the long term, given the aggravated N limitation at depth.