Beta-2 glycoprotein I (β2GPI) is the main antigenic target for antiphospholipid antibodies (aPL), the serological markers of antiphospholipid syndrome (APS). Domain I (DI) of β2GPI has lately been identified as the main epitope targeted by antibodies reacting against β2GPI. DI is a cryptic epitope, becoming available for autoantibody binding when β2GPI opens from a circular to a fish-hook configuration. Antibodies targeting β2GPI-DI are more frequently detected in patients with a full-blown syndrome than in asymptomatic aPL carriers or in patients with infectious diseases that have reactivity toward the whole molecule. Interestingly, anti-DI antibodies are strongly positively correlated with thrombotic and pregnancy manifestations, enabling identification of patients at higher risk of clinical events. However, available tests to detect anti-DI antibodies still lack standardization. Moreover, some APS patients develop antibodies reacting against β2GPI epitopes other than DI, suggesting that other anti-β2GPI antibody subsets may be clinically relevant. Available evidence on anti-DI antibodies in APS is herein critically reviewed.