Abstract: Objective：To explore the construction conditions and methods of lentivirus-mediated GDF-5 gene overexpressed rat adipose stem cells (ASCs-GDF-5). Methods：Rat ASCs were isolated and cultured using collagenase digestion method. The cell morphology was observed with the growth curve being tested. Besides, the cell phenotypes were identified. Lentiviral vector system with GDF-5/GFP chimeric gene was prepared and the infection efficiency was explored under series of MOI (1, 5, 10, 20, 40, 60, 80, 100). The optimal MOI was determined and the infection efficiency was tested using FCM. Highpurified ASCsGDF-5 were obtained through fluorescence activated cell sorting with FCM. The positive rate of infected cells was verified further with DAPI staining. The viability of infected cells was evaluated with CCK-8 assay. Results：Rat ASCs were successfully isolated and cultured. The markers (CD90, CD29, CD44, CD105) expressed in mesenchymal stem cell were positive in cultured cells. In contrast, the hematopoietic cell surface antigens (CD45, CD34) and bone marrow stem cell surface antigen (CD106) were negative. GDF-5 gene overexpressed lentiviral vector system was successfully constructed. The optimal MOI was 40, with the infection rate of 65%. The positive rate of infected cells was increased to 96% through fluorescence activated cell sorting using FCM. There was no significant difference in the viability and growth curve between infected and noninfected cells with CCK-8 assay. Conclusions：Rat ASCs can be cultured using collagenase digestion method. Without significant effect on cell viability, the positive rate of infected cells can be significantly increased through fluorescence activated cell sorting using FCM.