Background: During pregnancy, the maternal immune system undergoes dynamic adaptations to induce tolerance to the fetus that is effectively a tissue graft, while maintaining immune surveillance against pathogens. Placental extracellular vesicles (EVs), carry fetal antigens and when administered intravenously (i.v.), as occurs during pregnancy, may be involved in inducing maternal immune tolerance of the fetus. However, we have shown that intraperitoneal (i.p.) injection of pEVs in mice xenografted with ovarian tumours results in an antitumour immune response. Objectives: To identify differences in the interaction of pEVs with maternal immune cells following different routes of pEV administration. Methods: pEVs were isolated from human placental explants and labelled with CMPTX. Pregnant CD1 mice (gestation day 12.5) were injected i.v. (n=5) or i.p. (n=5) with 100 μg of pEVs. Twenty-four hours after injection, peripheral blood mononuclear cells (PBMCs) and splenocytes were isolated, stained and analysed by flow cytometry. Results: Significantly more EVs were associated with monocytes in the PBMCs after i.v. injection (p=0.0360) than following i.p. injection. In the spleen, i.v. injection resulted in more interactions of pEV with Tregs than i.p. injection (p<0.0001) possibly producing a more tolerogenic response. Additionally, i.v. injection resulted in more pEV-associations with total splenic macrophages (p=0.0008) and specific splenic macrophage subpopulations, CD169+ (p=0.0029) and MACRO+ macrophages (p<0.0001). Discussion: The interaction of pEVs with maternal immune cells depends upon the route of delivery. This may explain why pEVs are implicated in maternal immune tolerance in pregnancy but stimulate antitumour immune responses when delivered i.p.