Sunflower (Helianthus annuus) can potentially be used for uranium (U) phytoremediation. However, the influence of arbuscular mycorrhizal fungi (AMF) on key rhizosphere processes and plant U uptake remains insufficiently researched. We hypothesized that AMF inoculation could enhance sunflower tolerance to U stress by improving plant physiological performance and modifying rhizosphere properties. To test this hypothesis, this study examined the effects of AMF (Funneliformis mosseae, Glomus etunicatum, and their co-inoculation) on sunflowers under U stress, encompassing plant growth and physiological traits, rhizosphere properties, enzyme activities in the rhizosphere soil, uranium speciation in the rhizosphere soil, and the accumulation and distribution of uranium within the plant. Results showed that AMF successfully colonized the roots, enhancing plant growth, biomass, and gas exchange, while improving photosynthetic efficiency and reducing non-photochemical quenching. In the rhizosphere, AMF elevated soil respiration, organic matter, dissolved organic carbon, and microbial biomass carbon; improved phosphatases, urease, catalase, and sucrase activities; also reshaped U speciation, increasing exchangeable and carbonate-bound fractions while decreasing those bound to organic matter, Fe/Mn oxides, and residual phases. Moreover, AMF reduced U concentration in leaves and stems, promoted U retention in belowground tissues, and significantly lowered the U translocation factor. These findings demonstrate that AMF inoculation improves sunflower tolerance to U stress by enhancing physiological performance, modifying rhizosphere properties, and immobilizing U in roots, supporting its potential use in phytoremediation strategies for U-contaminated environments.