Abstract: Objective To construct and identify the retroviral vector liver-type pyruvate kinase（LPK） promoter with human insulin gene（hINSg） in gene or stem cell therapy for diabetes mellitus. Methods Parent（pMDN-SIN,p54） and pM54（LPKp-hINSg） plasmids were amplified,purified and identified with restriction enzyme. The pM54,p54 and pCMVβGal plasmids were transfected into pT67 and Phoenix E package cell lines with the FuGENE method. 3T3 cells served as controls. Supernatants were collected 48 or 72 hours after transfection with DNA. Seventy hours after 3T3 cells were infected,supernatants were collected. Transfection ratio was calculated with trypan blue staining,and INS expression was detected by ELISA. Results The retroviral vector pM54（LPKp-hINSg） was successfully constructed and transfected into pT67 and infected 3T3 cells. The human insulin gene expression was upregulated. Conclusion The retroviral vector pM54 we constructed lays a vital foundation for further study of gene therapy or gene therapy in combination with stem cells for diabetes mellitus.