Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important cytokine in the mammalian immune system. It has been expressed in Escherichia coli with the same biological activity as the native protein. Here, we report the synthesis of a murine recombinant GM-CSF in an E. coli cell-free protein synthesis system with a high yield. Since there are two disulfide bonds in the native structure of GM-CSF, an oxidizing redox potential of the reaction mixture was required. By pretreating the cell extract with iodoacetamide (IAM), the reducing activity of the cell extract was inactivated, and upon further application of an oxidized glutathione buffer, most of the synthesized GM-CSF was found in its oxidized form. However, the GM-CSF thus formed showed low activity because of poor folding. With the addition of DsbC, the periplasmic disulfide isomerase from E. coli, a high yield of active GM-CSF was produced in the cell-free reaction. Finally, successful folding of the cell-free synthesized GM-CSF-his6 was confirmed by its cell-proliferation activity after purification with a Ni2+ chelating column.