Vanadium pentoxide (V2O5) is the most stable phase among many transition metal vanadium oxides, and has already been widely used in many fields. In this study, the morphological, structural, and optical responses of V2O5 film to ultrafast laser irradiation was investigated. The third-order nonlinear optical properties of V2O5 film were measured by common Z-scan technique, and the results showed that V2O5 film has self-defocusing and saturable absorption characteristics. The third-order nonlinear absorption coefficient and nonlinear refractive index were calculated to be -338 cm/GW and -3.62 x 10(-12) cm(2)/W, respectively. The tunable saturated absorption with modulation depth ranging from 13.8% to 29.3% was realized through controlling the thickness of vanadium pentoxide film. V2O5 film was irradiated by ultrafast laser with variable pulse energy, and the morphological and structural responses of the V2O5 to the laser with different energy densities were investigated. The irreversible morphological and structural responses of V2O5 films to ultrafast laser irradiation was analyzed using the phase-contrast microscope and Raman spectrum. The chemical structure change from V2O5 to V6O13 was considered the main reason for refractive index modification.