Preeclampsia (PE) is a pregnancy-specific hypertensive disorder which affects ~8% of all pregnancies worldwide. The placenta is considered to be the main culprit in the etiology of PE as the symptoms of PE typically abate following removal of the placenta. PE is also known to be mediated in part by excessive maternal immune system activation and inflammation. We developed a mouse model of PE by excessively activating TLR3, which recognizes dsRNA from necrotic tissue and viruses; however, it is unknown whether TLR3 activation in maternal systemic cells, in paternally-derived placental trophoblasts, or both play a role in the development of hypertension during pregnancy. We hypothesized that both contribute and to test this we used the following mating schemes: 1) female WT mice mated with male WT mice (control), 2) female TLR3 KO mated with WT male (Ma TLR3 KO, TLR3 deficiency in all maternal cells), 3) male TLR3 KO mated with female WT (Pa TLR3 KO, TLR3 deficiency in paternally-derived trophoblasts, the major cell type in the placenta), and 4) female TLR3 KO mated with male TLR3 KO (MaPa TLR3 KO, TLR3 deficiency in all maternal cells and trophoblasts). Pregnant WT and TLR3 KO (Ma, Pa, and MaPa) mice were subjected to poly I:C or saline treatment on gestational days 13, 15, and 17. Poly I:C treatment induced pregnancy-dependent hypertension in pregnant WT mice (PPIC WT) (day 17 SBP in mmHg: PPIC WT: 1475 vs. P WT: 1004; p<0.05), however this was attenuated in PPIC Ma TLR3 KO mice (1092 mmHg; p<0.05 vs. P WT) and PPIC Pa TLR3 KO mice (1091 mmHg; p<0.05 vs. P WT), and completely normalized in PPIC MaPa TLR3 KO mice (1023 mmHg; P>0.05 vs. P WT). PPIC WT mice exhibited endothelial dysfunction, proteinuria, and increased fetal demise compared to P WT mice which were attenuated in PPIC Ma TLR3 KO and PPIC Pa TLR3 KO mice, but completely normalized in PPIC MaPa TLR3 KO mice. PPIC WT mice exhibited splenomegaly which was only completely reduced in PPIC MaPa TLR3 KO mice. In conclusion, our data suggest that both maternal systemic and paternal-derived placental trophoblast TLR3 activation contribute to the full development of PE-like symptoms in mice.