Comprehensive climate change mitigation necessitates soil carbon (C) storage in cultivated terrestrial ecosystems. Deep-rooted perennial crops may help to turn agricultural soils into efficient C sinks, especially in deeper soil layers. Here, we compared C allocation and potential stabilization to 150 cm depth from two functionally distinct deep-rooted perennials, i.e., lucerne (Medicago sativa L.) and intermediate wheatgrass (kernza; Thinopyrum intermedium), representing legume and non-legume crops, respectively. Belowground C input and stabilization was decoupled from nitrogen (N) fertilizer rate in kernza (100 and 200 kg mineral N ha−1), with no direct link between increasing mineral N fertilization, rhizodeposited C, and microbial C stabilization. Further, both crops displayed a high ability to bring C to deeper soil layers and remarkably, the N2-fixing lucerne showed greater potential to induce microbial C stabilization than the non-legume kernza. Lucerne stimulated greater microbial biomass and abundance of N cycling genes in rhizosphere soil, likely linked to greater amino acid rhizodeposition, hence underlining the importance of coupled C and N for microbial C stabilization efficiency. Inclusion of legumes in perennial cropping systems is not only key for improved productivity at low fertilizer N inputs, but also appears critical for enhancing soil C stabilization, in particular in N limited deep subsoils.