Soybean (Glycine max Merrill) seedlings were inoculated with arbuscular mycorrhizal (AM) fungi, Glomus mosseae, G. etunicatum, or/and soybean cyst nematode (SCN) Heterodera glycines in split-root systems. The dynamic changes of colonization of AM fungi, penetration rate of SCN, disease index, activities of peroxidase (POD), phenylalanine ammonia lyase (PAL), β-1,3-glucanase and chitinase in roots were analyzed. Both two arbuscular mycorrhizal (AM) fungi were able to decrease the disease index and penetration rate of SCN whether in one compartment or the other compartment of the split-root systems, the AM fungus and SCN inoculation in the same one compartment showed greater effectiveness than that in the two compartments. Compared with the treatment with SCN, the number of cysts in rhizospheric soil, cysts on roots and SCN in roots decreased by 47.4%, 58.9%, 46.6% and 50.5%, 67.0%, 57.5% respectively in the treatment inoculated with G. mosseae or G. etunicatum, and inoculated with SCN 3 weeks after AM fungal inoculation. It was indicated that AM fungi could significantly inhibit the development of nematode. The activities of the four enzymes in roots colonized by G. mosseae or G. etunicatum could be induced in a period of time. The AM fungus not only competed with SCN for colonization sites in one compartment, but also activated the defense mechanism of soybean, elicited the defensive enzymes in the other compartment. It was suggested that AM fungi can both systemically and locally antagonize SCN.