In vitro studies have documented β2 glycoprotein I (β2GPI) binding to endothelial cells (ECs) and trophoblast using antiphospholipid antibodies. The in vivo binding of β2GPI to these cells and the conditions that favor their interaction have not been investigated. We analyzed the in vivo distribution of cyanine 5.5-labeled β2GPI in mice and evaluated the effect of pregnancy and circulating antibodies on its tissue localization. The signal was detected in the liver by whole body scan and ex vivo analysis. The β2GPI failed to bind to the vascular endothelium and reacted only with the ECs of uterine vessels. In pregnant mice the protein was localized on ECs and trophoblast at the embryo implantation sites. Immunized mice showed a similar β2GPI biodistribution to naive mice but the immunized pregnant animals exhibited a significant increase in fetal loss associated with C3 and C9 deposition at the implantation sites. Treatment of mice with LPS after β2GPI-Cy5.5 injection promoted protein localization on gut and brain ECs associated with IgG, C1q, and C9 deposition in immunized mice. These findings indicate that β2GPI binding to EC requires priming with pro-inflammatory factors which is not needed for uterine and placental localization probably dependent on hormonal changes.