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 the individual 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 C2B decrease lipid order while C2A increases the lipid order. However, at higher protein concentrations a threshold is reached where, the effect on order is reduced or even reversed. The presence of 1% PIP2 in the lipid bilayer lowers these threshold concentrations. This change in lipid order is largely due to POPS and suggests that the effect on lipid order is due to the demixing of POPS. Interestingly, this effect is not seen in another negatively changed lipid POPG. The effect of synaptotagmin on demixing in PC:PS membrane bilayers was further investigated by fluorescence quenching. Our results show that the C2AB and C2B demix the PS in the presence of Ca2+, while C2A has little effect. Natural abundance 13-C HSQC NMR experiment on POPC:POPS (3:1) vesicles shows that the binding of synaptotagmin changes the chemical shift of PS in the presence of Ca2+, indicating an interaction between the protein and lipid. Taken together, these data suggest that synaptotagmin induces a PS-specific modulation of the acyl chain lipid order as a result of PS-demixing, which may play a role in the mechanism of Ca2+-mediated fusion in the central nervous system.