This work describes the production of (R,R)-2,3-butanediol in Escherichia coli using glycerol by metabolic engineering approaches. The introduction of a synthetic pathway converting pyruvate to (R,R)-2,3-butanediol into wild-type E. coli strain BW25113 led to the production of (R,R)-2,3-butanediol at a titer of 3.54 g/l and a yield of 0.131 g product/g glycerol (26.7 % of theoretical maximum) with acetate (around 3.00 g/l) as the dominant by-product. We therefore evaluated the impacts of deleting the genes ackA or/and poxB that are responsible for the major by-product, acetate. This increased production of (R,R)-2,3-butanediol to 9.54 g/l with a yield of 0.333 g product/g glycerol (68.0 % of theoretical maximum) in shake flask studies. The utilization of low-priced crude glycerol to produce value-added chemicals is of great significance to the economic viability of the biodiesel industry.