The present invention relates to a method of culturing mesenchymal stem cells using gamma-irradiated serum, and more particularly to a method for culturing mesenchymal stem cells, which can improve the adhesion and proliferation rate of stem cells using a medium containing gamma-irradiated serum and an antioxidant. The method for culturing mesenchymal stem cells according to the present invention can restore the adhesion and proliferation rate of stem cells when culturing the stem cells using gamma-irradiated FBS, which is safe from contamination sources but reduces the efficiency of adhesion and proliferation of the cells. Thus, the inventive method is useful for the production of stem cells for cell therapy.