Abstract: Objective To isolate and culture human amniotic fluid mesenchymal stem cells（HAFMSC）,observe their biological characteristics,and identify their multi-differentiation ability. Methods HAFMSC were isolated from amniotic fluid of the second trimester of gestation with the modified two-step method.Their proliferating ability was observed with the MTT method.Expressions of surface antigens including CD29,CD34,CD44,CD45,CD73,CD90,HLA-ABC and HLA-DR were detected by FACS.Expression of OCT-4 in HAFMSC at Nanog mRNA level was detected by RT-PCR.HAFMSC were induced with media of adipogenic,osteogenic and endothelial cells.Their differentiation ability was observed with oil red O,von Kossa and cellular immunofluorescence staining. Results The HAFMSC were adhered 2-3 days after cultured,and cell colonies were observed 10-15 days after cultured and arranged in a whirl-like form with a "S" shape of growth curve after passage 3.FACS showed that the CD29,CD44,CD73,CD90 and HLA-ABC were positively expressed while the CD34,CD45 and HLA-DR were negatively expressed.RT-PCR demonstrated that the OCT-4 and Nanog mRNA were positively expressed in HAFMSC.Twenty-one days after differentiation was induced with adipogenic and osteogenic cells,the HAFMSC were positively stained with oil red O and cellular immunofluorescence staining displayed that the vWF antibody was positively expressed 4 weeks after differentiation was induced with osteogenic cells. Conclusion HAFMSC can be isolated from amniotic fluid and are characterized by mesenchymal stem cells with a multi-differentiation potential.