Orthorhombic V₂O₅ nanowires were successfully synthesized via a hydrothermal method. A cell-configuration system was built utilizing V₂O₅ as the cathode and 1 M Mg(ClO₄)₂ electrolyte within acetonitrile, together with Mg_xMo₆S₈ (x ≈ 2) as the anode to investigate the structural evolution and oxidation state and local structural changes of V₂O₅. The V₂O₅ nanowires deliver an initial discharge/charge capacity of 103 mAh g^–1/110 mAh g^–1 and the highest discharge capacity of 130 mAh g^–1 in the sixth cycle at C/20 rate in the cell-configuration system. In operando synchrotron diffraction and in operando X-ray absorption spectroscopy together with ex situ Raman and X-ray photoelectron spectroscopy reveal the reversibility of magnesium insertion/extraction and provide information on the crystal structure evolution and changes of the oxidation states during cycling.