Although start-up is a critical procedure in biofilm-based processes, systematic research aimed at improving start-up efficiency in hydrogen-based membrane biofilm reactors (H2-MBfRs) is lacking. In this study, we investigated the effects of H2 pressure on the stabilized denitrification performance of H2-MBfRs during start-up. Following inoculation with anoxic sludge, the biomass was acclimated to various H2 pressures. The increase in denitrifying microbial activity under high H2 pressures (i.e., H2 flux) shortened the denitrification lag time and reduced NO3–N removal flux, indicating an improved acclimation efficiency. The acclimated biomass was attached to membranes under different H2 pressures. High H2 pressures impeded biomass attachment, possibly because of bubble-less evaporation. Reducing the H2 pressure appeared to be necessary for improving the biomass attachment. However, this measure decreased previously acclimated denitrification activity. Therefore, the loss of both acclimation and biomass adhesion must be considered during the attachment phase. The highest removal flux (0.63 e- eq/m2-d) was obtained after just 3.5 d of acclimated biomass attachment (i.e., activated sludge floc) at 1.6 bar. During Phase 3, the initial performance was further improved to 0.89 e- eq/m2-d by gradually increasing the H2 pressure. These results provide a start-up strategy for improving the initial performance of H2-MBfRs.