Preeclampsia is a common pregnancy-specific disorder characterized by elevated blood pressure and proteinuria. Activation of the maternal immune system and impaired placental angiogenesis are thought to contribute to the pathogenesis of preeclampsia. TLR9 (Toll-like receptor 9) plays a role in innate immunity, defending the organism against infection. The purpose of this study was to determine whether TLR9 inhibits angiogenesis at the fetomaternal interface under conditions of preeclampsia. We confirmed the downregulation of VEGFA (vascular endothelial growth factor A) and upregulation of TLR9 and sFLT1 (soluble vascular endothelial growth factor receptor 1) in placentas from preeclamptic women. Then, we established a mouse model with preeclampsia-like symptoms using the synthetic TLR9 agonist CpG (cytidine–phosphate–guanosine)-ODN (oligodeoxynucleotide; ODN1826). We observed the downregulation of VEGFA and the upregulation of sFLT1 in placentas from the preeclampsia-like animal model and in trophoblasts treated with CpG-ODN (ODN2006). In addition, silencing TLR9 promoted the migration and invasion of HTR8/SVneo cells. In conclusion, TLR9 is capable of robustly suppressing angiogenesis by differentially regulating the expression of VEGFA and sFLT1 at the fetomaternal interface, potentially contributing to the development of preeclampsia.