Abstract Soil organic matter (SOM) stocks contain nearly three times as much carbon (C) as the atmosphere and changes in soil C stocks may have a major impact on future atmospheric carbon dioxide concentrations and climate. Over the past two decades, much research has been devoted to examining the influence of warming on SOM decomposition in topsoil. Most SOM, however, is old and stored in subsoil. The fate of subsoil SOM under future warming remains highly uncertain. Here, by combining a long-term field warming experiment and a meta-analysis study, we showed that warming significantly increased SOM decomposition in subsoil. We also showed that a decade of warming promoted decomposition of subsoil SOM with turnover times of decades to millennia in a tall grass prairie and this effect was largely associated with shifts in the functional gene structure of microbial communities. By coupling stable isotope probing with metagenomics, we found that microbial communities in warmed soils possessed a higher relative abundance of key functional genes involved in the degradation of organic materials with varying recalcitrance than those in control soils. These findings suggest warming may considerably alter the stability of the vast pool of old SOM in subsoil, contributing to the long-term positive feedback between the C cycle and climate.