Poly-β-hydroxybutyrate (PHB) was produced by recombinant E. coli HMS174 in a glucose-mineral medium. The medium composition and culture conditions were optimized by a series of shake flask experiments. The optimal initial concentrations of glucose and (NH4) 2SO4 were about 20 g/L and 4 g/L, respectively. The optimal temperature, pH, culture and inoculum volume were 37, 7.5, 25% and 10%, respectively. The final cell dry weight, PHB dry weight, PHB content and glucose conversion efficiency reached 8.06 g/L, 6.58 g/L, 81.64% and 98.56%, respectively, at 23.67 g/L initial glucose concentration after 48 h culturing. Different fed-batch cultures were also carried out in the shake flask.