CD4(+) regulatory T lymphocytes (Treg cells) play a crucial role in maintaining the normal immune homeostasis. Foxp3, as a key marker for Treg cells, is widely used to identify Treg cells, not only in humans but also in other species, like mouse, porcine, ovine, and bovine. To detect reproducible Treg cells is important for evaluating the state of the immune system, and thus, it is necessary to optimize Foxp3 staining. Here, we present a comparative study of MF23 and FJK-16s clones of anti-mouse Foxp3 antibodies, used in combination with two different fixation/permeabilization buffers. For Foxp3 staining, the fixation/permeabilization buffer and Foxp3 antibody FJK-16s clone from eBioscience were better than those from BD Pharmingen, with the best fluorochrome PE. Moreover, when using the best combination, there was a highly significant positive correlation between CD25(+) T cells and CD25(+)Foxp3(+) T cells. Therefore, the CD25 marker can be used as an alternative to the Foxp3 antibody. As FJK-16s is also applicable for detecting bovine, porcine, canine, ovine, and equine Foxp3 antibodies, these results will be helpful not only in quantifying the frequencies of mouse Treg cells, but also in accurately detecting Treg cells of the other species mentioned above by multicolor flow cytometry.