V2O5 thin films were deposited with different precursor concentrations of 0.01, 0.05, and 0.1 M on glass substrates by spray pyrolysis technique, then the optimized films were annealed in different ambients (air, oxygen, and vacuum). The results showed that by increasing the concentration, the films grew along the (001) direction with an orthorhombic structure. Field emission scanning electron microscopy showed that nanorods were formed when depositing 0.05 molar of VCl3. We conclude that with the precursor concentration, the surface nanostructure can be well-controlled. Annealing improved the crystallinity under all ambients, but the best crystallinity was achieved in vacuum. It was revealed that the as-deposited films had the highest transmission, whereas the films annealed in air had the lowest. When annealed in air, the optical band gap decreased from 2.45 to 2.32 eV. The sheet resistance, resistivity, mobility, conductivity, and carrier concentration were measured for all the prepared V2O5 films.