V2O5 thin films were prepared by pulsed laser deposition (PLD) over a wide substrate temperature range, 30-500 C, and were characterized by studying their microstructure and optical properties. Rutherford backscattering spectrometry (RBS), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), and UV-vis-NIR spectral measurements were made on the PLD V2O5 films to understand the effect of substrate temperature on the chemical composition, elemental distribution, surface morphology, and optical properties. The substrate temperature strongly influences the structure and optical properties of PLD V2O5 films and a correlation exists between the growth conditions, grain structure, and optical characteristics. The grain size increased, associated with a change in surface morphology, with increasing substrate temperature. The optical energy band gap of PLD V2O5 films is strongly dependent on the substrate temperature and decreased from 2.47 to 2.12 eV with the increase in temperature from 30 to 500 C.