Our aim was to determine the effect of soil characteristics and root traits on soil aggregate stability at an inter- and intra-site scale in a range of agro-ecosystems. We also evaluated the effect of soil depth and the type of land use on aggregate stability. Soil aggregate stability, soil physicochemical properties and fine root traits were measured along land use gradients (from monocultures to agroforestry systems and forests), at two soil depths at four sites (Mediterranean and tropical climates) with contrasting soils (Andosol, Ferralsol, Leptosol and Fluvisol). Aggregate stability was much lower in deep than in surface soil layers, likely linked to lower soil organic carbon (SOC) and lower root mass density (RMD). Locally, and consistently in all sites, land use intensification degrades soil aggregate stability, mainly in surface soil layers. Soil organic carbon, cation exchange capacity and root traits: water-soluble compounds, lignin and medium root length proportion were the most important drivers of aggregate stability at the inter-site level, whereas SOC, root mass and root length densities (RMD, RLD) were the main drivers at the intra-site level. Overall, the data suggest different controls on soil aggregate stability globally (soil) and locally (roots). Conversion from forests to agricultural land will likely lead to greater C losses through a loss of aggregate stability and increased soil erosion.