To gain more insight into the interactions between vanadium and L-proline, the dynamic transformation of the reaction system of vanadium with L-proline and the coordination structures of the vanadium-containing products in 0.15 mol/L NaCl ionic medium mimicking physiological conditions were explored by multinuclear (H-1, C-13, V-51) NMR, ESR, ESI-MS as well as density functional theory (DFT) calculations. Spectroscopic evidence and computational results showed that a monoperoxovanadium species [VO(O-2)(L-proline)(2)](-) was a major product, where L-proline coordinated to vanadium via nitrogen and oxygen atoms in a bidentate manner to form a distorted pentagonal bipyramidal structure. The species [VO(O-2)(L-proline)(2)](-) underwent chemical changes in solution at room temperature, finally leading to the reduction of vanadium(V) to vanadium(IV) and the formation of [VO(L-proline)(2)]. In the tetrahedral structure of the reduction product [VO(L-proline)(2)], L-proline also coordinated to vanadium in a bid ciliate manner. Such an investigation may be helpful for a better understanding of vanadium complexes as insulin-enhancing agents for the treatment of diabetes.