Quantitative characterization of the strength of peripheral membrane protein-lipid bilayer interactions is fundamental in the understanding of many protein targeting pathways. SecA is a peripheral membrane protein that plays a central role in translocating precursor proteins across the inner membrane of E. coli. The membrane binding activity of the extreme N-terminus of SecA is critical for translocase function. Yet, the mechanical strength of the interaction and the kinetic pathways that this segment of SecA experiences when in proximity of an E. coli polar lipid bilayer has not been characterized. We directly measured the N-terminal SecA-lipid bilayer interaction using precision single molecule atomic force microscope (AFM)-based dynamic force spectroscopy. To provide conformational data inaccessible to AFM, we also performed all-atom molecular dynamics simulations and circular dichroism measurements. The N-terminal 10 amino acids of SecA have little secondary structure when bound to zwitterionic lipid head groups, but secondary structure, which rigidifies the lipid-bound protein segment, emerges when negatively charged lipids are present. Analysis of the single molecule protein-lipid dissociation data converged to a well-defined lipid-bound-state lifetime in the absence of force, τ0lipid = 0.9 s, which is well separated from and longer than the fundamental time scale of the secretion process, defined as the time required to translocate a single amino acid residue (∼50 ms). This value of τ0lipid is likely to represent a lower limit of the in vivo membrane-bound lifetime due to factors including the minimal system employed here.