Vanadium oxide thin films were prepared by DC reactive magnetron sputtering at substrate temperatures ranged from room temperature to 400 °C, and the effects of substrate temperature on microstructure, optical, and electrical properties of the films were analyzed. XRD spectra showed that the main component of the thin films was V2O5 with orthorhombic structure. Tensile stresses exist in the thin films. With increasing substrate temperature, the films gradually changed from amorphous to polycrystalline in nature. High peak transmittance of about 80% for the samples prepared at temperatures less than 300 °C is observed. The optical bandgap of the thin films slightly decreased from 2.51 to 2.44 eV at first, and then increased with increasing substrate temperature. The optical bandgap changes can be attributed to grain size increasing and structural modification of thin films with substrate temperature. All samples had a large sheet resistance of about 106–107 Ω/□ and negative temperature coefficient of resistance (TCR) of about −1.0% K−1. The sheet resistance decreased as the deposition temperature increased, and the TCR presented an initial decrease and then an increasing trend.