Soil represents an important environmental compartment that can be regarded as a final sink for metal nanoparticles including silver particles (Ag-NPs). Assessing realistic exposure scenarios, including the bioavailability of Ag-NPs for soil organisms, requires taking into account that Ag-NPs can undergo physico-chemical transformations, such as sulphidisation, before interacting with organisms. However, differentiating between uptake of true metal NPs and that of released ions is essential to assess the actual role of these two metal forms in toxicity over time. The present study quantified the toxicokinetic rate constants of particulate and ionic Ag in Eisenia fetida exposed to soil treated with pristine Ag-NPs (50 nm), Ag2S-NPs (20 nm) as an environmentally relevant form, and AgNO3 as an ionic control. Results showed that the uptake and elimination rate constants of Ag in earthworms exposed to Ag-NPs and AgNO3 were not significantly different from each other, whereas the uptake of Ag2S-NPs was significantly lower. Interestingly, the biogenic formation of particulate Ag (∼10% of the total Ag accumulated over time) in earthworms exposed to AgNO3 led to a kinetic pattern of particulate Ag similar to that of pristine Ag-NPs. SEM-EDX analysis confirmed the presence of particulate Ag in earthworms exposed to both Ag-NPs and AgNO3, showing that these particles were different from those to which earthworms were exposed. We demonstrated that around 85% of the Ag accumulated in the worms after exposure to Ag-NPs and AgNO3 was present as ions or as particles with size <20 nm. Additionally, the low accumulation of the non-soluble, sulphidised form of nano-Ag, reflecting aged particles in the environment, confirms the importance of ionic uptake of Ag. This study clearly shows that the main form of Ag taken up in earthworms is the ionic species, which stresses the fundamental need to use environmentally relevant forms of metal NPs in performing ecotoxicological tests, because pristine NPs may behave completely differently.