The performances of betaine on the metabolic processes of vitamin B12-producing Pseudomonas denitrificans were investigated in this paper. The results showed that betaine was an indispensable methyl-group donor for vitamin B12 biosynthesis, but large amounts of the extracellular glycine accompanied by betaine metabolism would impose a severe restriction on the cell growth of P. denitrificans. By further using a comparative metabolomics approach coupled with intracellular free amino acids analysis for the fermentation processes with betaine addition (10 g/l) or not, it was found that betaine could highly strengthen the formation of some key precursors and intermediates facilitating vitamin B12 biosynthesis, such as δ-aminolevulinic acid (ALA, the first precursor of vitamin B12), glutamate (an intermediate of ALA via C5 pathway), glycine (an intermediate of ALA via C4 pathway), and methionine (directly participating in the methylation reaction involved in vitamin B12 biosynthetic pathway). Therefore, the performances of betaine on P. denitrificans metabolic processes were not only serving as a decisive methyl-group donor for vitamin B12 biosynthesis, but also playing a powerfully promoting role in the generation of vitamin B12 precursors and intermediates.