<p>Both NpO₂²⁺ and NpO₂⁺ exist as linear dioxo-cations in aqueous solutions, but they show dramatically different extraction behavior by tributylphosphate (TBP). The mechanism underlying this difference remains unclear. In this work, the complexation of TBP with NpO₂²⁺ and NpO₂⁺ in organic solutions is detected by spectrophotometric and microcalorimetric titrations and theoretical calculations. NpO₂²⁺ can form 1:1 and 1:2 complexes with TBP, and the stability constants decrease with the increase in water content from 0.5% to 1.5%. In contrast, NpO₂⁺ can only form a 1:1 complex with TBP at 0.5% water content and no complexation is observed at higher water content. The enthalpies and entropies of the complexation of TBP with NpO₂²⁺ and NpO₂⁺ at 0.5% water content are obtained by microcalorimetric titrations, indicating the formation of inner-sphere Np(VI)-TBP complex and outer-sphere Np(V)-TBP complex. Based on the experimental results, the structures of the extracted complexes and their extraction energies are obtained by density functional theory (DFT) calculations. The extraction energies for Np(VI) and Np(V) by TBP are positive and negative, respectively, consistent with the extraction behavior of the neptunyl ions by TBP. Results in this work provide structural and energetic insights into the mechanism for the differences in the extraction of Np(VI, V) by TBP. Extractant that can remove the water molecules from the hydrated NpO₂⁺ ion should be selected to extract NpO₂⁺ into the organic phase.</p> Graphical Abstract <p></p>