Amniotic fluid-derived mesenchymal stem cells (AF-MSC) are newly described, excellent seed cells that have good differentiation capability and are convenient to obtain. However, it is important to develop a method to isolate and culture AF-MSC efficiently. Amniotic fluid samples were obtained from rabbits and the adherence method was used for AF-MSC culture. Flow cytometry, western blot, and immunofluorescence studies were used to analyze the phenotypic characteristics of the cultured AF-MSC. Amniotic fluid-derived mesenchymal stem cells were successfully isolated and cultured from amniotic fluid. Flow cytometric analysis demonstrated that these cells expressed CD29 and CD44, while they did not express CD34. The expression of transcription factor Oct-4 was confirmed by western blot and immunofluorescence analysis. Using the adherence method, we developed a successful, reproducible protocol for the isolation of AF-MSC from amniotic fluid. The results of our phenotypic analysis revealed that the AF-MSC isolated in the present study were multipotent cells.