Abstract: The metabolic regulation of inorganic nitrogen sources involved in vitamin B12 shake-flask fermentation by Pseudomonas denitrificans was studied in this paper.Firstly, the effects of various inorganic nitrogen sources ( (NH4) 2SO4 、NH4NO3 、NaNO3 、 (NH4) 2HPO4 and NH4Cl) on P.denitrificans metabolism were investigated, and the result showed that ammonium sulfate was more favorable for vitamin B12 fermentation.Based on this fact, the effects of NH4+ concentrations on P.denitrificans fermentation processes were further researched, and it was discovered that the presence of high NH4+ concentration could significantly accelerate the cell growth but had negative action on vitamin B12 biosynthesis.Therefore, in order to overcome the contradiction between cell growth and vitamin B12 biosynthesis, the strategy of NH4+ restrictively feeding was implemented in shake-flask fermentation by P.denitrificans.