Hierarchical and porous V2O5 microspheres have been fabricated by a refluxing approach followed by annealing in air. The resulting porous V2O5 microspheres typically have diameters of 3–6 μm and are constructed of intertwined laminar nanocrystals or crosslinked nanobricks. It is found that the vanadyl glycolates rinsed with water have pronounced pore structures than that rinsed with ethanol alone. In addition, the configuration of the vanadyl glycolates microspheres can be tuned during the refluxing along with stirring. The possible formation processes of the vanadyl glycolates and V2O5 products have been discussed based on the experimental data. Electrochemical tests indicate that the hierarchical and porous V2O5 microspheres exhibit relatively high and stable Li+ storage properties. The porous V2O5 microspheres assembled by intertwined nanoparticles maintain reversible Li+ storage capacities of 102 and 80 mAh g−1, respectively; whilst the porous V2O5 microspheres assembled by crosslinked nanobricks maintain reversible Li+ storage capacities of 100 and 85 mAh g−1 over 100 cycles at current rates of 0.5 and 1 C, respectively. The superior Li+ storage performance of the hierarchical and porous V2O5 microspheres could mainly be ascribed to the improved electrode/electrolyte interface, reduced Li+ diffusion paths, and relieved volume variation during lithiation and delithiation processes.