We used X-ray reflectivity to investigate the structures of phospholipid multilayers with transcription-activating-factor-derived peptide (TDP) as a function of the membrane charge density. Mixed phospholipid multilayers of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl -sn-glycero-3-phosphoserine (DPPS) with different mixing ratios (C:S) were used to elucidate the various charge densities in a plasma membrane. We fixed the peptide/lipid molar ratio (P/L) and varied the DPPC/DPPS molar ratio in the mixed multilayer. In the pure DPPC multilayer, the incorporation of TDP had nearly no effect on the bilayer thickness of the mixed lipid multilayer. However, in the mixed DPPC/DPPS multilayer, the incorporation of TDP decreased the bilayer thickness, suggesting that the TDP peptide had a stronger interaction with DPPS than with DPPC and caused disorder in the lamellar structure. Combining this with the refined X-ray reflectivity (XR) data, we concluded that the TDP existed more in the headgroup region of the TDP-induced segregated DPPS in the mixed multilayer and caused significant membrane thinning.