Abstract: It has been proved that artificial chaperone-assisted protein refolding can promote the refolding of chemically denatured proteins via the sequential addition of low molecular weight “artificial chaperones”. The aim of the present study was to compare three methods for refolding recombinant Chicken IL-18 protein (rChIL-18) expressed in E. coli. The recombinant chicken IL-18 protein was efficiently expressed in inclusion bodies in the E. coli expression system. The inclusion bodies were thoroughly denatured with 6 M of Guanidine Hydrochloride (GH) and refolded by using the methods of artificial chaperone-assisted refolding, GH-deionized water diafiltration and GH-glutathione renaturation. Results of our comparative studies demonstrated that refolding yield of rChIL-18 was at least two times higher using the method of artificial chaperone-assisted refolding (42.45%) than those using GH-deionized water diafiltration (10.67%) and GH-glutathione renaturation (14.83%). The optimal SI values were observed at different concentrations of rChIL-18 proteins refolded by the methods of artificial chaperone-assisted refolding (150 μg L^-1), GH-deionized water diafiltration (600 μg L^-1 and GH-glutathione renaturation (400 μg L^-1). In conclusion, the method of artificial chaperone-assisted refolding is more efficient for the refolding of recombinant chicken IL-18 than those of GH-glutathione renaturation and GH-glutathione renaturation.