Abstract: Objective To establish a parabiosis model between allogenic conspecific adult mice to study two-way paradigm. Methods Fifty-four female Balb/c mice and 54 male C57BL/6 mice were paired and equally divided into 3 groups, namely group 1 with normal saline (NS) injection, group 2 with injections of spleen cells and cyclophosphamide (CP), and group 3 injected with spleen cells, CP, and cyclosporin A (CsA). The treatments were performed by injecting the spleen cells from one of the mice in a pair into the other via tail vein and vise versa, and two days after the operation, CP (150 mg/kg) was injected intraperitoneally. Intraperitoneal CsA (30 mg/kg daily) injection was given starting from 2 days before till 7 days after the operation. Twelve of the 18 pairs of parabiosis mice in each group were separated after 1 week, and part of the skin were transplanted to each other. The maintenance of parabiosis was observed in the other 6 pairs of parabiosis mice were observed. Mixed lymphocyte reaction (MLR) and delayed-type hypersensitivity (DTH) were observed and studied in the separated mice. Result The duration of parabiosis maintenance and skin survival of the group 3 was significantly longer than those in the other two groups, and group 3 showed suppressed MLR and DTH. Conclusion With the application of immunosuppressants, we have successfully established the two-way paradigm model in mice.