Regulatory B (Breg) cells are a special subset of immunoregulatory cells with unique phenotypes and functions. In this study, human CD19(+)CD25(high) Breg cells were purified from human peripheral blood. Based on the coculture system of Breg cells and CD4(+) T cells in vitro, Breg cells were found to promote the increase in regulatory T (Treg) cells while decreasing the number of Th17 cells. Breg cells regulate Treg cells through two processes: cell-cell contact and cytokines. TGF-beta sRII, a blocker of transforming growth factor-beta (TGF-beta), can attenuate the effects of Treg elevation, suggesting that TGF-beta is the main cytokine, while Breg cells rather than interleukin-10 (IL-10) regulate the differentiation of Treg cells. However, Th17 cells were mainly regulated by cytokines, without an obvious regulatory effect on cell-cell contacts. Breg cells may regulate Th17 cells by a pathway independent of TGF-beta and IL-6. The coculture of Breg cells and CD4(+) T cells led to changes in the cytokine spectrum, which included significant increases in IL-4, IL-6 and IL-10 but not obvious changes in IL-2, IFN-gamma and TNF. The inhibitory effect of Breg cells was weakened by blocking cell-cell contacts in cultures separated with the Transwell chamber because IL-10 decreased while IL-6 increased when compared with co-cultured Breg and CD4(+) T cells. When the IL-10 inhibitor IL-10sR alpha was added, IL-6 and TNF levels significantly increased, while treatment with the TGF-beta inhibitor TGF-beta sRII did not result in similar changes, suggesting that IL-10 is an important molecule to inhibit the proinflammatory factors IL-6 and TNF in this culture system.