Apolipoprotein H (ApoH), also known as β2-glycoprotein I, is a plasma glycoprotein with its in vivo physiological and pathogenic roles being closely related to its interaction with negatively charged membranes. Although the threedimensional crystal structure of ApoH has been recently solved, direct evidence about the spatial state of ApoH on the membrane is still lacking. In this work, the interactions of ApoH with the lipid layer are studied by a combination of lipid monolayer approach and surface concentration determination. The spatial state of the orientation of ApoH on the lipid layer is investigated by analyzing the process of membrane-attached ApoH molecules being extruded out from the phospholipid monolayer by compression. The results show that on neutral lipid layer ApoH has an upright orientation, which is not sensitive to the phase state of the lipid layer. However, on acidic lipid layer, ApoH may have two forms of orientation. One is an upright orientation in the liquid phase region, and the other is flat orientation on the condensed domain region. The variation of the spatial state of ApoH on the lipid layer may relate to a variety of its physiological functions.