Purpose Rhizosphere processes are known to modify uptake of elements from soil, but limited information is available for hyperaccumulators. We investigated labile Ni fractions and their kinetics of replenishment in the rhizospheres of the Ni-hyperaccumulator Odontarrhena serpyliffolia, the Ni-excluder Holcus lanatus and in bulk soils collected at the same serpentine outcrop. Methods Labile Ni fractions in rhizosphere and bulk soil were evaluated using conventional extractions and also by the Diffusive Gradients in Thin Films (DGT) technique. DGT data were used to predict the kinetics of Ni resupply using DIFS modelling. Chemical imaging of Ni distribution along roots using DGT coupled with laser-ablation was conducted. Results Labile Ni forms were higher in both rhizosphere than in bulk soils, together with an increase in dissolved organic C, cation exchange capacity and the Ca/Mg ratio. Ni fractionation indicated a shift towards less stable Ni fractions in the rhizosphere, particularly in the hyperaccumulator. DIFS modelling showed that the rhizosphere of the excluder was able to sustain the initially lower soluble Ni concentration through replenishment from the solid phase, while Ni resupply in the rhizosphere of the hyperaccumulator was not sufficient to maintain the initially high concentrations of soluble Ni. However, the amount of DGT-labile Ni was higher in the rhizosphere of the hyperaccumulator compared to the excluder in all deployment times. Conclusion Our data suggest that compounds derived from root activity, in particular DOC, are important controls of Ni availability to plants growing on serpentine soil.