The controllable synthesis of large-area and uniform hexagonal boron nitride (h-BN) films has been recently achieved on metal-boron alloy catalysts with the use of N2 feedstock, representing important progress in an economic and environmentally friendly process. However, the systematic investigation of the growth mechanism is still lacking, which impedes the further development of this method. In this work, on the basis of density functional theory (DFT) calculations and experiments, we reveal the vacancy-assisted growth mechanism of h-BN on Fe2B substrate. It is found that B vacancies created by the formation of BN dimers play important roles in the migration of B and N atoms near the catalyst surface. The diffusions of B and N atoms in the Fe2B substrate need to overcome energy barriers of only less than 1.5 eV, which enables abundant dissolution of N atoms near the catalytic surface. Moreover, we found the critical barrier for h-BN growth is in the nucleation stage, which is ∼2 eV. These advantages enable the synthesis of h-BN at a low temperature of 700 K in our experiments. This vacancy-assisted growth of h-BN films on Fe2B substrates is beneficial to the wafer-scale fabrication of multilayer materials, paving the way to potential applications in two-dimensional electronic and optoelectronic devices.