The release of carbon (C) emissions into the atmosphere is the primary driver of global climate change. Addressing this is the biggest environmental challenge faced by humankind. To overcome the challenge, a growing focus has been on the largest terrestrial C store, soil, for its ability to sequester further C from the atmosphere. Due to the intensity of agricultural soil management, agricultural soils have a large C deficit that can be filled yielding various co-benefits. The scale and feasibility of enhancing this C sink, however, is much debated. Recently, soil beneath the topsoil (i.e. subsoil) has been proposed as a better potential target than topsoil. This is because the conditions, soil characteristics and low disturbance allows C in agricultural subsoils to reach thousands of years of age. The overall aims of this thesis were to 1) evaluate and investigate subsoil-targeted C sequestration strategies; and 2) investigate the mechanisms underpinning subsoil greenhouse gas (GHG) dynamics (production and consumption) to understand how these impact C sequestration success. Firstly, I conducted an extensive review and meta-analysis of the literature describing mechanisms of subsoil C stabilisation to better evaluate subsoil-targeted C sequestration strategies and explore opportunities and limitations of this field, concluding that the strategies can offer more potential to sequester C in the long-term, but this is highly context dependent. Secondly, a series of laboratory incubations was conducted to test an approach to enhance subsoil C sequestration via the addition of iron to enhance C stabilisation. Despite the reduction in microbial C respiration of specific C forms, bulk soil C was not protected in the soil tested, so the method was not deemed an effective strategy for this soil. Next, a deep rooting grass field trial with or without root excluding mesh buried at different depths was established. Measuring GHGs above and below the mesh and using the concentration gradient method (CGM), more C was respired from root-accessible soil though this made no difference at the soil surface. This suggests that more C is gained than is lost from deeper rooting. To address the second aim, GHGs were measured at different depths across 2 growing seasons. The CGM was tested for gas flux estimation of carbon dioxide (CO2) to assess the method across different conditions and understand the movement and contribution of the gas to the surface-atmosphere flux. The CGM performed poorly in drought conditions and evidence of depth-dependent GHG consumption was found. Finally, an incubation study in a precision-controlled environment with added 15N2O was conducted to disentangle the biological and physical mechanisms underpinning N2O production and consumption in soil. The diffusion rates did not differ with depth, but deeper soil consumed more N2O when drier due to aerobic denitrification, suggesting subsoils have high denitrification potential despite the low microbial biomass. Together, this research provides a valuable contribution to the understanding of the behaviour of C and GHGs in the subsoil environment, which is essential to pursuing subsoil C sequestration – a useful tool for aiding climate change mitigation. Going forward, greater evidence and policy support is required for large-scale adoption of subsoil-targeted C sequestration strategies.