Sum frequency generation (SFG) vibrational spectroscopy was used to study surface immobilization effects on the interactions between antimicrobial peptide cecropin P1 (CP1) and model cell membranes. While free CP1 in solution interacted with a model cell membrane composed of a phosphatidylglycerol (PG) bilayer, electrostatic interaction led to the attachment of CP1 molecules onto the PG surface and the hydrophobic domain in the lipid bilayer enabled the peptides to insert into the bilayer and form alpha-helices from random coil structures. While CP1 molecules immobilized on a self-assembled monolayer interacted with PG lipid vesicles, the intensity of the SFG peak for the peptide alpha-helix decreased as the PG vesicle concentration increased. It was believed that when surface-immobilized CP1 molecules interacted with lipid vesicles, they lay down on the surface or became random coils. When the immobilized CP1 interacted with a PG lipid monolayer on water, the strong interaction led to the lying-down orientation of all of the surface-immobilized peptides as well. Differently, no significant interactions between surface-immobilized CP1 with the mammalian cell membrane model 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer were observed. Our results suggest that, instead of membrane insertion, the electrostatic interactions between the surface cationic charges of CP1 and anionic bacterial membranes may play an important role in the antimicrobial activity of the surface-immobilized CP1 peptide.