Glomalin-related soil protein (GRSP) is an essential bioactive component that may respond to heavy metal stress; however, its exact influence on metal bioavailability and the associated mechanism remains poorly understood. This study investigated the speciation and distribution of heavy metals in soil aggregates associated with GRSP through macroscopic and microscopic approaches. A field study showed that the metal ions were distributed to the macro-aggregate fraction by partitioning the particle size classes during phytoremediation. Partial least squares path modeling (PLS-PM) demonstrated that the heavy metal bioavailability was negatively affected by aggregate stability (61.5%) and GRSP content (52.8%), suggesting that the soil aggregate properties regarding GRSP were vital drivers in mitigating environmental risk closely associated with toxic metal migration in soil–plant systems. The nonideal competitive adsorption (NICA)-Donnan model fitting suggested that GRSP were rich in acid site density, and the complexation with deprotonated groups dominated the speciation of heavy metals in soil. Further, the microfocus X-ray absorption/fluorescence spectroscopy analysis indicated that GRSP might promote the formation of stable metal species by binding with sulfur-containing sites. This study highlights the role of GRSP in heavy metal sequestration in contaminated soils, providing new guidance on the GRSP intervention for phytoremediation strategies. • Soil aggregate and GRSP were vital in mitigating bioavailability of toxic metals. • GRSP contributed to metal binding by partitioning to the macro-aggregate fractions. • Stable metal speciation was linked with sulphur-containing sites on GRSP. • Metal stabilization regulated by GRSP provided new guidance for phytoremediation.