Abstract: Although betaine is one of the crucial precursors in vitamin B12 (VB12) fermentation by Pseudomonas denitrifican, the cell growth will be inhibited at its high concentration. So it is important to keep the betaine concentration under suitable level for high VB12 production. The effects of betaine of different concentrations on the VB12 production were investigated with different feeding strategies. The results indicated that the extracellular release of VB12 could be stimulated by maintaining a low betaine concentration, and it also showed that the low betaine concentration could significantly enhance the accumulation of VB12 especially in the late fermentation. The highest VB12 production could be achieved when the betaine concentration was kept at 3.0 g/L in the 50 L fermenter, which was 129% of that at 9.0 g/L.