Our recent study has demonstrated induction of regulatory B cells (Bregs) by bacterial DNA in MRL/lpr mice leading to attenuation of SLE. This suggests a regulatory role for the gut bacteria in SLE development. However, the mechanism by which bacterial DNA induces Breg cell differentiation remains to be elucidated. Bacterial DNA contains unmethylated CpG motifs which are recognized by the innate immune molecule, toll-like receptor 9 (TLR9). We therefore hypothesize that gut bacteria-derived DNA induces Bregs in a TLR9-dependent manner, which in turn protects against lupus initiation. To test our hypothesis, TLR9 global knockout (Tlr9 ^-/-) MRL/lpr mice and their heterozygous (Tlr9 ^+/-) littermates were randomized into 2 groups per genotype. Beginning at 4 weeks of age, one group in each genotype received 80 µg E. coli double- stranded DNA (dsDNA) once a week by oral gavage for 4 consecutive weeks, whereas the other group received phosphate buffered saline (PBS) as control. Urine samples were collected before euthanasia whereas blood, spleen, lymph nodes, and kidneys were collected and processed following euthanasia at 15 weeks of age. In vitro culture treatment with bacterial DNA was used to investigate a TLR9-dependent, B cell-intrinsic or -extrinsic mechanism in Breg cell induction. Our data showed a trend in decrease of proteinuria, as well as sizes of spleen and lymph nodes, with bacterial DNA treatment in Tlr9 ^+/- mice. Interestingly, serum levels of anti-dsDNA IgM, which has been shown to be protective against lupus, were significantly increased in bacterial DNA-treated Tlr9 ^+/- mice, although the pathogenic anti-dsDNA IgG remained unchanged. Additionally, flow cytometry analysis of splenocytes showed an increasing trend in the percentage of interleukin-10 positive (IL-10⁺) Bregs in bacterial DNA-treated Tlr9 ^+/- mice and a concomitant decrease in double negative (DN, or CD4^-CD8^-) T cells. In the kidney, bacterial DNA treatment also decreased the percentage of CD45⁺ cells in Tlr9 ^+/- mice. Importantly, all bacterial DNA- induced changes observed in Tlr9 ^+/- MRL/lpr mice, including those of proteinuria, organ weight, anti-dsDNA IgM, splenic Bregs, splenic DN T cells and renal CD45⁺ cells, were abrogated in Tlr9 ^-/- MRL/lpr mice. Bacterial DNA treatment of cocultures of B and non-B cells from Tlr9 ^+/+ and Tlr9 ^-/- MRL/lpr mice revealed a TLR9-dependent, but B cell-extrinsic, induction of Bregs mediated by IL-6 produced most likely from myeloid cells. Altogether, our study suggests possible attenuation of SLE by gut bacteria-derived DNA through a B cell-extrinsic, TLR9-dependent mechanism that involves the induction of IL-10⁺ Bregs and secretion of potentially protective anti- dsDNA IgM.