Cell membrane fusion is a fundamental biological process involving in a number of cellular living functions. Regarding this, divalent cations can induce fusion of the lipid bilayers through binding and bridging of divalent cations to the charged lipids, thus leading to the cell membrane fusion. However, the elaborate mechanism of cell membrane fusion induced by divalent cations is still needed to be elucidated. Here, surface/interface sensitive sum frequency generation vibrational spectroscopy (SFG-VS) and dynamic light scattering (DLS) were applied in this research to study the responses of phospholipid monolayer to the exposure of divalent metal ionsi.e. Ca2+ and Mg2+. According to the particle size distribution results measured by DLS experiments, it was found that Ca2+ could induce inter-vesicular fusion while Mg2+ could not. An Octadecyltrichlorosilane self-assembled monolayer (OTS-SAMs)/lipid monolayer system was designed to model the cell membrane for the SFG-VS experiment. We found, Ca2+ can interact with the lipid PO2- head groups more strongly, resulting in cell membrane fusion more easily, in comparison to Mg2+. No specific interaction between the two metal cations and the C=O groups was observed. However, the C=O orientations changed more after Ca2+ -PO2- binding than Mg2+ mediation on lipid monolayer. Meanwhile, Ca2+ can induce dehydration of the lipids (which should be related to the strong Ca2+ - PO2- interaction), leading to the reduced hindrance for cell membrane fusion. .