Synaptotagmin 1 (syt1) functions as the Ca(2+) sensor in neuronal exocytosis, and it has been proposed to act by modulating lipid bilayer curvature. Here we examine the effect of the two C2 domains (C2A and C2B) of syt1 on membrane lipid order and lateral organization. In mixtures of phosphatidylcholine and phosphatidylserine (PS), attenuated total internal reflection Fourier transform infrared spectroscopy indicates that a fragment containing both domains (C2AB) or C2B alone disorders the lipid acyl chains, whereas the C2A domain has little effect upon chain order. Two observations suggest that these changes reflect a demixing of PS. First, the changes in acyl chain order are reversed at higher protein concentration; second, selective lipid deuteration demonstrates that the changes in lipid order are associated only with the PS component of the bilayer. Independent evidence for lipid demixing is obtained from fluorescence self-quenching of labeled lipid and from natural abundance (13)C NMR, where heteronuclear single quantum correlation spectra reveal Ca(2+)-dependent chemical shift changes for PS, but not for phosphatidylcholine, in the presence of the syt1 C2 domains. The ability of syt1 to demix PS is observed in a range of lipid mixtures that includes cholesterol, phosphatidylethanolamine, and varied PS content. These data suggest that syt1 might facilitate SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors)-mediated membrane fusion by phase separating PS, a process that is expected to locally buckle bilayers and disorder lipids due to the curvature tendencies of PS.