The dependence of the solution structure of neamine on pH was determined by NMR and AMBER molecular dynamics methods at pD 3.3, pD 6.5, and pD 7.4 in D 2O at 25 °C. Unlike neamine structures at pD 3.3 and 6.5, which essentially showed only one conformer, slowly exchanging primary, P-state, and secondary, S-state, neamine conformers populated on the NMR time scale at ∼80% and ∼20%, respectively, were detected at pD 7.4 with kinetic constants k on(P→S) = 1.9771 s −1 and k off(S→P) = 1.1319 s −1. A tertiary, T-state, neamine species populated at ∼3% was also detected by NMR at pD 7.4. The p Ka values determined by NMR titration experiments are p Ka1 6.44 ± 0.13 for N3 of ring-II, p Ka2 7.23 ± 0.09 for N2′ of ring-I, p Ka3 7.77 ± 0.19 for N1 of ring-II, and p Ka4 8.08 ± 0.15 for N6′ of ring-I. Ring-I and ring-II of the P-state neamine and ring-I of the S and T-states of neamine possess the 4C 1 chair conformation between pD 3.3 and pD = 7.4. In contrast, ring-II of the S and T-states of neamine most likely adopt the 6rH 1 half-chair conformation. The P and S-states of neamine exhibit a negative syn-ψ glycosidic geometry. The exocyclic aminomethyl group of ring-I adopts the gt exocyclic rotamer conformation around physiological pHs while the gg exocyclic rotamer conformation predominates in acidic solutions near and below pH 4.5. Neamine exists in the P-state as a mixture of tetra-/tri-/di-protonated species between pD 4.5 and pD 7.4, while the S-state neamine exist only in a di-protonated species around physiological pDs. The existence of the S-state neamine may facilitate binding of neamine-like aminoglycosides by favorable entropy of binding to the A-site of 16S ribosomal RNA, suggesting that novel aminoglycoside compounds carrying a S-state neamine pharmacophore can be developed.