Subtropical forests certainly contribute to terrestrial global carbon storage, but we have limited understanding about the relative amounts and of the drivers of above-ground biomass (AGB) variation in their region. Here we assess the spatial distribution and drivers of AGB in 119 sites across the South American subtropical forests. We applied a structural equation modelling approach to test the causal relationships between AGB and environmental (climate and soil), structural (proportion of large-sized trees) and community (functional and species diversity and composition) variables. The AGB on subtropical forests is on average 246 Mg ha−1. Biomass stocks were driven directly by temperature annual range and the proportion of large-sized trees, whilst soil texture, community mean leaf nitrogen content and functional diversity had no predictive power. Temperature annual range had a negative effect on AGB, indicating that communities under strong thermal amplitude across the year tend to accumulate less AGB. The positive effect of large-sized trees indicates that mature forests are playing a key role in the long-term persistence of carbon storage, as these large trees account for 64% of total biomass stored in these forests. Our study reinforces the importance of structurally complex subtropical forest remnants for maximising carbon storage, especially facing future climatic changes predicted for the region.