Cell-penetrating peptides (CPPs) can translocate across cell membranes, and thus have great potential for the cellular delivery of macromolecular cargoes. However, the mechanism of this cellular uptake process is not yet fully understood. In this study, time-lapse light sheet optical microscopy was implemented to obtain a parallel visualization of the translocating dynamics of individual human immunodeficiency virus 1 (HIV-1) transactivator of transcription (Tat) peptide conjugated quantum dots in complex cellular terrains. Remarkable trajectory aggregates were observed on the cell surface, possibly caused by interaction between the Tat peptides and heparan sulfate groups on the plasma membrane. Spectral-embedding analysis of the trajectory aggregates revealed a manifold formed by isotropic diffusion and directed movement. Further stochastic analysis indicated that the Tat peptides may have remodeled the actin framework in the cytoplasm to reduce their interaction with local membrane environment. The membrane deformation induced by Tat-peptide attachment increased with the disruption of the actin framework, yielding higher interactions on the Tat-coated particle. Characteristic hot spots for interaction were detected on the membrane, suggesting that a funnel passage had formed for the Tat-coated particles. This finding offers valuable insight into the cellular delivery of nanoscale cargoes, suggesting an avenue for direct therapeutic delivery.