Peyer's patches (PPs) play an important role in the induction of immune responses in the intestine, but regulation of Toll-like receptor (TLR)-induced innate immune responses in PPs is not well understood. We investigated the responses of PPs and other immune cells to the TLR9 agonist, CpG oligodeoxynucleotide (ODN). Peripheral blood mononuclear cells and lymph node cells secreted significant amounts of interferon (IFN)-alpha, IFNgamma, and interleukin (IL)-12 following stimulation with CpG ODN. In contrast, PP cells exhibited poor cytokine responses, despite abundant expression of TLR9 mRNA. PP cells spontaneously secreted high levels of IL-10, and the primary source of the IL-10 was resting CD5(-)CD11c(-)CD21(+) B cells. Neutralization of the IL-10 or depletion of CD21(+) B cells resulted in a significant increase in CpG-induced IFNalpha-response in PPs, suggesting that IL-10 from B cells regulate innate responses in PPs. These IL-10-secreting PP B cells may represent a novel subset of the recently proposed regulatory B cells (B(regs)) in the intestine.