Magnesium-based materials offer a promising, cost-effective, and high-capacity solution for hydrogen storage. However, their slow kinetics and elevated absorption/desorption temperatures limit their large-scale application. In this study, a series of Mg1-x/Vx (x = 0.05, 0.10, 0.15, 0.20) multilayer films were designed and fabricated using an ultra-high vacuum magnetron sputtering technique. Transmission electron microscopy (TEM) analysis revealed the presence of a discontinuous nanoscale V single-phase interlayer between the Mg layers, confirming the successful formation of Mg/V multilayer films. The surface of the deposited films predominantly consists of hexagonal particles of varying sizes stacked upon one another. Upon hydrogenation, tetragonal rutile structures of MgH2 and V2H were formed. The Mg0.95V0.05 film absorbed 6.40 wt% hydrogen within 15 min at 150 °C and desorbed 0.34 wt% hydrogen within 1.8 h at the same temperature, with an initial dehydrogenation temperature of 79 °C. The activation energies for hydrogen absorption and desorption were estimated to be 60 ± 7 and 140 ± 10 kJ/mol H2, respectively, significantly lower than the corresponding values of 100 and 160 kJ/mol H2 for conventional pure Mg/MgH2. The enhanced hydrogen absorption performance was attributed to the catalytic effect of nanocrystalline V at the Mg/V incoherent interface, while the improved desorption performance was due to the synergistic catalytic effect of nanocrystalline V2H at the MgH2/V2H incoherent interface. DFT calculations indicated that the interaction at the Mg/V incoherent interface promoted H atom adsorption and diffusion, significantly enhancing the hydrogenation and dehydrogenation performance of the multilayer films.