It was the first time to study the catalytic effect of mesoporous magnetic Fe3O4 nanoparticles on the biohydrogen production. The mixed culture used in this study just suffered from an alkaline shock and lost its bioactivity of hydrogen production. We use mesoporous Fe3O4 nanoparticles and ferrous ions as activators to recover the bioactivity of the mixed culture. The results indicate that the improvement of biohydrogen yield by mesoporous Fe3O4 was obvious larger than that by ferrous ions. The maximum yield of cumulative hydrogen production was obtained at the mesoporous Fe3O4 nanoparticles of 400 mg·L-1, which is 26% higher than that of the blank. The lag phases for hydrogen production in the tests added with mesoporous Fe3O4 nanoparticles were decreased to 12 h, which are 50 h less than those of the corresponding ferrous ions and blank tests.