Subsoils are characterized by low concentrations of organic carbon (OC). Nevertheless, they contain more than half of the global soil OC because of their large volume. This discrepancy suggests that subsoils might further sequester carbon (C), thus acting as potential sinks for atmospheric C. Plant roots and associated rhizodeposits are a major OC input source to subsoils. However, whether and how increased OC inputs via plant roots to subsoils affect soil C sequestration mechanisms remains unclear. Here we set up a pot experiment with European Beech (Fagus sylvatica L.) seedlings to investigate the effect of tree roots and associated rhizosphere development on soil aggregation and C allocation in topsoil vs. subsoil material collected from three forest sites of different parent materials. Over a 5-month growth period, the seedlings developed a dense root system transforming the whole soil volume into root-affected (i.e., rhizosphere) soil. We found that roots and the associated rhizosphere development increased the amount of macroaggregates in the two finest-textured subsoils. The most C-poor and fine-textured subsoil had a 15% increase in bulk OC concentration, indicating a potential for C sequestration in subsoils by enhanced macroaggregation. Across subsoils, rooting strongly enhanced microbial abundance and was especially correlated with fungal abundance and a shift in the fungal-to-bacterial- ratio. The strong fungal growth was likely the cause for the enhanced macroaggregation in these subsoils. In topsoils, however, rooting treatment decreased macroaggregate abundance, potentially through the disruption of preexisting aggregates, as indicated by the concomitant increase in microaggregates. Our study supports the growing awareness that OC dynamics may be governed by different mechanisms in top- and subsoils, respectively. It demonstrates that the enhanced addition of OM via plant roots to subsoils boosts fungal growth and thereby increases macroaggregate formation, potentially facilitating C sequestration by occlusion.