Toll-like receptor 9 (TLR9) enhances proinflammatory responses, but whether it can act in a regulatory capacity remains to be established. In experimental murine AKI induced by cisplatin, Tlr9-/- mice developed enhanced renal injury and exhibited fewer intrarenal regulatory T cells (Tregs) compared with genetically intact mice. A series of reconstitution and depletion studies defined a role for TLR9 in maintaining Treg-mediated homeostasis in cisplatin-induced AKI. When Rag1-/- mice were reconstituted with nonregulatory CD25- splenocytes from wild-type (WT) or Tlr9-/- mice, AKI was similarly enhanced. However, when Rag1-/- mice were reconstituted with CD4+CD25+ regulatory cells, WT CD4+CD25+ cells were more renoprotective and localized to the kidney more efficiently than Tlr9-/- CD4+CD25+ cells. In Treg-depleted Foxp3DTR mice, reconstitution with naive WT CD4+CD25+ cells resulted in less severe AKI than did reconstitution with Tlr9-/- Tregs. Tlr9-/- mice were not deficient in CD4+CD25+ cells, and WT and TLR9-deficient Tregs had similar suppressive function ex vivo. However, expression of adhesion molecules important in Treg trafficking was reduced on peripheral CD4+CD25+ cells from Tlr9-/- mice. In conclusion, we identified a pathway by which TLR9 promotes renal Treg accumulation in AKI.