By using atomic force microscopy (AFM), we clearly show that the antimicrobial peptide affects the molecular interaction between lipopolysaccharide (LPS) and immune proteins (lipopolysaccharide binding protein [LBP] and CD14). To reconstruct an in vivo interaction, LBP and LPS (the Ra, Rc, and Re forms from Salmonella minnesota, with varying lengths of the saccharide region) were immobilized onto the AFM tip using a chemical spacer linker. We examined the interaction between the proteins on the tip and model lipid bilayer biomembranes including CD 14, in both the presence and absence of the antimicrobial peptide, polymyxin B (PMB). When LPS was present, the binding force between the LBP-LPS complex and CD 14 increased dramatically, compared to that seen between LBP and CD 14 alone. Longer LPS saccharide regions resulted in higher binding forces. The data suggest that LPS may have an important influence on the binding of LBP to CD 14 and that the saccharide region of LPS is influential in this regard. It was also found that the antimicrobial peptide PMB, at or above a particular concentration, specifically inhibited the binding between LBP-LPS and CD14.