Ash fusibility is closely related to ash slagging, which has significant impact on the clean and efficient utilization of petroleum coke (petcoke). Some mineral elements in petcoke, especially vanadium (V), are considered to be responsible for ash related slagging during thermal conversion of petcoke. This study investigates the effect of vanadium pentoxide (V2O5) on synthetic petcoke ash fusibility from different perspectives, including V2O5 content variation, temperature rising, and atmosphere change. X-ray diffraction (XRD) and scanning electronic microscopy (SEM) were used to determine the mineral transformation and surface morphology of ash and slag at a high temperature. The ash-melting process was simulated by thermodynamic equilibrium calculations via a multicomponent system. The results show that ash fusion temperatures (AFTs) of synthetic ash samples vary markedly with the V2O5 content increasing. Moreover, AFTs exhibit a significant difference between reducing and oxidizing atmospheres, which can be ascribed to the different transformation behaviors of minerals in ash under different atmospheres. With the V2O5 content increasing, the V-containing species formed under a reducing atmosphere are vanadium trioxide (V2O3) and coulsonite (FeV2O4). Both can contribute to the progressive increase of AFTs because of their high melting points. However, AFTs under an oxidizing atmosphere initially decrease and then increase slowly. When the addition of V2O5 is below 25%, low-melting calcium pyrovanadate (Ca2V2O7) formed under an oxidizing atmosphere decreases the AFTs. When the V2O5 content continues to increase, the formations of high-melting spinel NiAl2O4 and V-bearing amorphous phase lead to the increase of AFTs.