Objective: In this paper we compared the two methods of cell sorting (magnetic cell sorting and flow cytometry sorting) for the isolation and function analysis of mouse CD4+ CD25+ regulatory T (Treg) cells, in order to inform further studies in Treg cell function. Methods: We separately used magnetic cell sorting and flow cytometry sorting to identify CD4+ CD25+ Treg cells. After magnetic cell separation, we further used flow cytometry to analyze the purity of CD4+ CD25+ Treg cells, trypan blue staining to detect cell viability, and propidium iodide (PI) staining to assess the cell viability. We detected the immune inhibition of CD4+ CD25+ Treg cells in the in vitro proliferation experiments. Results: The results showed that compared to flow cytometry sorting, magnetic cell sorting took more time and effort, but fewer live cells were obtained than with flow cytometry sorting. The CD4+ CD25+ Treg cells, however, obtained with both methods have similar immunosuppressive capacities. Conclusion: The result suggests that both methods can be used in isolating CD4+ CD25+ Treg cells, and one can select the best method according to specific needs and availability of the methodologies. Objective: In this paper we compared the two methods of cell sorting (magnetic cell sorting and flow cytometry sorting) for the isolation and function analysis of mouse CD4 + CD25 + regulatory T (Treg) cells, in order to inform further studies in Treg cell function. Methods: We separately used magnetic cell sorting and flow cytometry sorting to identify CD4 + CD25 + Treg cells. After magnetic cell separation, we further used flow cytometry to analyze the purity of CD4 + CD25 + Treg cells, trypan blue staining to detect cell viability, and propidium iodide We detected the immune inhibition of CD4 + CD25 + Treg cells in the in vitro proliferation experiments. Results: The results showed that compared to flow cytometry sorting, magnetic cell sorting took more time and effort, but fewer live cells were obtained with flow cytometry sorting. The CD4 + CD25 + Treg cells, however, obtained with similar methods for immunosuppressive capacities. Concl usion: The result suggests that both both can be used in isolating CD4 + CD25 + Treg cells, and one can select the best method according to specific needs and availability of the methodologies.