An Escherichia coli (E. coli) strain was engineered to synthesize 2-butanone from glucose by extending the 2,3-butanediol synthesis reaction sequence catalyzed by exogenous enzymes. To convert 2,3-butanediol to 2-butanone, B12-dependent glycerol dehydratase from Klebsiella pneumoniae was introduced into E. coli. It has been proposed that the enzyme has a weak activity toward 2,3-butanediol. The activity in E. coli is confirmed in this study. Furthermore, co-expressing coenzyme B12 reactivators increased the 2-butanone titer. This demonstration of 2-butanone production by extending the 2,3-butanediol biosynthetic pathway provides the possibility to produce this valuable chemical renewably.