The perturbation of lipid acyl chain order by fusion proteins is widely reported in the membrane of viral entry and fertilization process. Synaptotagmin is the Ca2+ trigger for membrane fusion in neuronal exocytosis, and it may act by modulating lipid packing or membrane curvature strain. The effects of soluble synaptotagmin (C2AB) and separate C2 domains (C2A and C2B) on the lipid order of POPC:POPS (3:1) membrane bilayer were examined with attenuated total reflection Fourier transformed infrared spectroscopy (ATR-FTIR). Our results show that C2AB and more noticeably C2B decrease the lipid order and C2A increases the lipid order in low concentrations. However, in concentrations higher than certain threshold values, the effects reduce or even reverse. The presence of 1% PIP2 in the lipid bilayer lowers these threshold concentrations. The role of Ca++ is ambiguous: Ca enhances the perturbation effect in presence of PIP2, and reduces the effect in absence of PIP2. Experiments with membrane bilayers composed of deuterated POPC and normal POPS indicate that the change in lipid order are largely due to POPS. These data suggest that lipid demixing and membrane curvature strain may play a role in the mechanisms of Ca2±mediated fusion in the central nervous system.