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Deep soil horizons represent a major but still poorly understood component of the soil carbon pool, despite their important contribution to long-term carbon storage and climate regulation. While carbon dynamics in topsoils have been extensively studied, much less is known about the mechanisms controlling carbon processing and stabilization at depth, particularly the role of microbial functioning and soil structure. This study investigated how microbial carbon use efficiency (CUE) varies with depth in a temporary agricultural grassland soil profile (10, 30 and 60 cm) under temperate conditions in Lusignan, France. Measurements were performed in both bulk soil and biostructures. Microbial CUE was estimated using two independent approaches (13C-based CUE and 18O–H₂O-based CUE), while microbial functional diversity was characterized using MicroResp and organic matter quality using Rock-Eval pyrolysis. Results showed contrasting depth-related patterns depending on the method used. 13C-based CUE increased with depth, with consistently higher values in biostructures than in bulk soil. In contrast, 18O-based CUE declined along the soil profile. Organic matter became progressively more stable and chemically mature with depth, while microbial communities shifted towards assemblages adapted to lower substrate availability and higher organic matter complexity. Variations in soil physical and chemical properties, organic matter quantity and quality, and microbial community structure therefore strongly depended on depth and the presence of biostructures, and jointly controlled microbial efficiency. These findings show that carbon storage in deep soil horizons depends strongly on microbial efficiency, soil structure and organic matter quality, and can be enhanced by management practices that increase carbon inputs and promote biostructure formation.Keywords: Subsoil carbon storage, Deep soil horizons, Land management practices, Microbial communities