BACKGROUND: There are two different routes for vitamin B-12 biosynthesis, which results in discrepancies and uncertainties of the dissolved oxygen (DO) concentration for vitamin B-12 fermentation. In this paper, the DO control strategy was explored for industrial vitamin B-12 fermentation by Pesudomonas denitrificans in 120000-L fermenter. RESULTS: A DO-stat strategy was first successfully scaled up from a 9000 L fermenter to a 120 000 L fermenter. Then a multi-stage DO control strategy was further established in the 120 000 L fermenter, in which the DO level was shifted from 810% (2048 h) to 25% (49106 h) and below 2% (107168 h) by gradually reducing the rate of aeration and agitation. As a result, 198.80 mg L-1 of vitamin B-12 was obtained, which was significantly higher than those obtained under the fermentations with one-stage DO control. CONCLUSIONS: The comparatively low DO level was favorable for vitamin B-12 biosynthesis, but it would have an extremely negative effect on cell growth. Compared with the low DO level maintained at all times of the fermentation process, a multi-stage DO control strategy could not only increase the biomass but also improve vitamin B-12 biosynthesis. Copyright (c) 2012 Society of Chemical Industry