Butyrate was produced in recombinant Escherichia coli strains by applying metabolic engineering strategies. The genes for producing butyrate were cloned from Clostridium acetobutylicum and then expressed in E. coli. To study important factors for improving the productivity of butyrate, we deleted pta and ptsG genes in E. coli and compared the effects of these gene deletions in E. coli B and K strains. The effect of carbon sources, glucose and glycerol, was also compared. A significant improvement of butyrate production was made when glycerol was used as a carbon source, resulting in 0.56 g/l of butyrate in LB medium with 1% (v/v) glycerol.