Plasmalogens (Pls) are widely distributed in the biological membrane of animals and certain anaerobic bacteria, but their functions in the cell membrane are still poorly understood. Decrease of phosphatidylethanolamine plasmalogen (PEPls) in the brain tissue of patients with Alzheimer's disease prompted us to investigate the effect of the membrane phosphorus lipid composition on the activity of γ-secretase that produces amyloid-beta protein (Aβ). To clarify the effect of phospholipids, including PEPls, on Aβ production, γ-secretase activity was measured in an in vitro assay using yeast microsomes and reconstituted liposomes. The presence of ethanolamine phospholipids in the proteoliposome weakened γ-secretase activity. In addition, increased PEPls content in total ethanolamine phospholipids further decreased the enzyme activity, indicating that γ-secretase activity is affected by the membrane phospholipid PEPls/PE ratio. Furthermore, PEPls from anaerobic bacterial cell membrane induced the same effect on γ-secretase activity.