With time-resolved high-precision single-particle tracking methodologies, we explored the adsorption and thermal motion of transacting activator of transcription (TAT) peptide-modified nanocargo on a model lipid bilayer in the nonelectrostatic domain. We found that the lateral and rotational motion of TAT peptide-modified nanocargo could be effectively suppressed on the surface of neutral lipid membrane, a feature that cannot be explained by existing hypotheses. A semiquantitative association activation energy analysis revealed that multiple weak bonds were required for the initial adsorption process. As a result, the localized multiple TAT peptides on the surface of the nanocargo can provide a pathway for the creation of a net of peptide-lipid complexes (e.g., lipid domain). The dragging forces caused by these complexes effectively confined the thermal motion of the nanocargo on the fluid membrane that cannot be achieved by individual peptides with random spatial and conformational distributions. These interesting findings could provide insightful information for the better understanding of the intracellular internalization mechanism of TAT peptide-modified nanocargo and might shed new light on the development of highly efficient intracellular carriers for site-specific delivery of drugs and genes.