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Objective: To establish a stable and high efficient method for collection of CD4+CD25+ regulatory T cells from rats in vitro. Methods: CD4+CD25+ regulatory T cells were isolated from the rat splenic cells through two steps by magic cell sorting (MACS) system. The first step was negative selection of CD4+ T cells by cocktail antibodies and anti-IgG magic microbeads, and the second step was positive selection of CD25+ T cells by anti-CD25 PE and anti-PE magic microbeads. The purity and viability of separated cells were measured by flow cytometry (FACS) and Trypan blue staining. The suppressive ability of seperated cells on the proliferation of CD4+CD25- T cells was assessed by cell proliferation assay. Results: The purity of negatively enriched CD4+ T cells was 79%-87% (83.6% ± 2.5%) , and the purity of positively enriched CD4+CD25+ T cells was 86%-93% ( 90.2 ± 1.8%) with the viability of 92%-95% (92.8% ± 3.4%). The enriched cells significantly suppressed the proliferation of CD4+CD25- T cells in mixed lymphocyte culture (P ＜ 0.05). Conclusion: An effective method can be established for enrichment of CD4+CD25+ regulatory T cells in two steps by MACS, with satisfied cell purity, viability and function.