Foxp3- IL-10-producing type I regulatory T (Tr1) cell is one of the helper T (Th) subsets that has strong immunosuppressive properties and helps to control excessive inflammatory responses such as tissue inflammation or autoimmunity. Thus, it is important to clarify the molecular mechanism of Tr1 differentiation for the regulation of the inflammatory responses, but it has not characterized yet. In this time, we investigated the regulation of Tr1 differentiation, especially focused on the role of phosphoinositide 3-kinase (PI3K), a lipid kinase that generates phosphatidylinositol triphosphate to transduce the cell activity. CD25+ naturally occurring regulatory T (nTreg)-depleted conventional CD4+ T cells were isolated from BALB/c WT mice, and were stimulated with anti-CD3ε and anti-CD28 Abs adding IL-27 in the presence or absence of PI3K inhibitors. In addition, to understand the role of PI3K pathway on Tr1 differentiation in vivo, PI3K inhibitor was administered in the differentiation of Tr1 cells in mice by anti-CD3ε antibody inoculation. We found that the suppression of the PI3K-Akt pathway results in impairment of IL-27–induced Tr1 (IL-27–Tr1) cell differentiation in vitro and in vivo. When the gene expression of Tr1-related molecules were examined by RT-PCR, the inhibition of PI3K activity down-regulated IL-21 receptor expression, followed by suppression of IL-10 expression in IL-27–Tr1 cells. These results suggest that the PI3K pathway enhances IL-10 expression by IL-27–Tr1 cells through up-regulation of IL-21 receptors.