BACKGROUND: Obstetric antiphospholipid syndrome (APS) is defined by pregnancy complications associated with antiphospholipid antibodies (aPL). The mechanisms of the pathogenic effects of aPL in pregnancy are poorly understood. Toll-like receptors (TLR) have been implicated previously in APS. OBJECTIVES: The aims of our study were (1) to determine aPL effects on trophoblastic cell fusion and differentiation, (2) to identify which TLR is involved in this process, and (3) to evaluate the efficacy of hydroxychloroquine (HCQ) to counteract the effects of aPL. METHODS: BeWo cells are a model for trophoblast fusion and differentiation. Fusion index was assessed by immunocytochemical examination, and biochemical differentiation by using ELISA-measured β-human choronic gonadotropin hormone (β-hCG) secretion. We used three types of aPL to study their effect on cell fusion and differentiation: aPL derived from obstetric APS patients and affinity purified and polyclonal rabbit anti-β2-glycoprotein-1 (anti-β2GP1) antibodies. Experiments on fusion were confirmed using primary cytotrophoblastic cells. RESULTS: All of the types of aPL used decreased the fusion index in BeWo and primary trophoblastic cells (64%, 52%, and 41% for BeWo cells and 67% and 62% for primary cells, respectively), and anti-β2GP1 antibodies decreased hCG secretion in BeWo cells (41%). To block TLR4 antibodies or to abolish TLR4 cell surface expression restored fusion index in both cell types and β-human choronic gonadotropin hormone excretion in BeWo cells. HCQ treatment induced the same effect and decreased TLR4 mRNA (40% and 35%, respectively) and protein expressions (62% and 42%, respectively) in BeWo cells. CONCLUSION: Anti-β2GP1 antibodies decrease trophoblastic differentiation via TLR4. This effect is restored by HCQ, suggesting its therapeutic interest in APS pregnancies.