The main goal of the current study was cloning and expression of human insulin gene in pichia pastoris expression system using genetic engineering techniques and the study of its treatment application. Total RNA of Recombinant human insulin were purified from fresh normal human pancreatic tissue of a brain death pa­tient Recombinant human insulin constructive expression vector was  transformed into Pichia pastoris GS115 by using electroporation method. The bioassay of the recombinant insulin was also determined in this study against induced hyperglycemic wistar rats .The results illustrated a highly significant (P< 0.005)  reduction in glucose levels of diabetic rabbits groups which were treated with recombinant insulin and commercially available standard human insulin, in comparison with non-treated animals, the recombinant insulin was found highly significant (P< 0.005) in reduction of  the blood glucose level of diabetic rats groups which were treated with recombinant insulin in comparison with untreated diabetic rats group. Also in this work the MTT assay and hemolysis test were carried out to evaluate the in vitro cytotoxicity of recombinant human insulin against fresh human blood cells. The results demonstrate that all tested concentrations of recombinant human insulin showed no any hemolytic activity and no inhibitory effect on the metabolism activity and cell viability of the human blood.