Objective To establish an orthotopic xenograft model of human glioma miniature pigs by treatment with immunosuppressive agents, and provide ideal experimental tools for screening glioma drug and imaging studies. Methods Human glioma cells U87-MG were transplanted into nude mice subcutaneously, and tumors were visible after two weeks. Then, we choose miniature pigs to be treated with oral immunosuppressant both cyclosporine soft capsules (20 mg / kg) and tacrolimus capsules ( 0. 01 mg / kg). Three days later, the fresh tumor tissue derived from nude mice were collected and trim them into 1 mm³ tissue pieces. The tumor tissue were transplanted into cerebral striatum of the miniature pigs treated with immunosuppressive agents through a stereotactic instrument. After 21 days of continuous use of immunosuppressive agents, the animals displayed obviously thin, and space-occupying lesions in the brain were detected by Magnetic Resonance Imaging(MRI)to confirm tumors formation. These miniature pigs were sacrificed and dissected, then the tumor tissues were fixed. The histomorphological characteristics of tumor were confirmed by H&E and IHC staining. Results Three weeks after orthotopic transplantation of the glioma tissue into the brain striatum of miniature pigs, 12 pigs have been implanted with U87-MG, 11 have presented a macroscopic significant tumor, with radiological and pathological characteristics of high-grade glioma. we found that the tumor incidence reached more than 90% by MRI scan. Both the HE staining result and growth characteristics of the tissue were consistent with tumor characteristics. The staining of human mitochondrial antibody demonstrated that this tumor was human-derived tissue and the glioma-specific marker GFAP was expressed positively. Conclusions Combined with cyclosporine soft capsule and tacrolimus immunosuppressive treatment, an orthotopic xenograft miniature pig model of human glioma was successfully established by transplanting tumor tissue.