This research examined factors which influence the expression of Human Growth hormone in periplasmic space of five recombinant Escherichia coli strains with different genetic structure under the transcriptional control different promoters. Selection of best strain was carried out based on three factors: growth rate, plasmid stability and expression level. Selected strain used for optimization experiments which discuss conditions for maximizing the growth and production of Human growth hormone in shake flask experiments and batch fermentation. Statistical experimental design was used for screening of main factors in production of periplasmic human growth hormone in shake flask experiments. The important factors considered were fermentation temperature, concentration of IPTG as Inducer, Time after Induction and culture optical density at Induction time. Information about the effect of different factors on rhGH production was investigated by using the two level fractional factorial designs. all of the experimental variables found to be significant. The most important factor in production of human growth hormone was time after induction. we selected our medium based on buffering capacity, specific growth rate and quality of medium. Using glucose as carbon source we observed glucose inhibition of human growth hormone expression. Using glycerol as carbon source we could overcome this limitation. In batch fermentations in bioreactor we reached to 68mg/L overall productivity.