The improvement of nonviral gene therapies relies to a large extent on understanding many fundamental physical and biological properties of these systems. This includes interactions of synthetic delivery systems with the cell and mechanisms of trafficking delivery vehicles, which remain poorly understood on both the extra- and intracellular levels. In this study, the mechanisms of cellular internalization and trafficking of polymer-based nanoparticle complexes consisting of polycations and nucleic acids, termed polyplexes, have been observed in detail at the cellular level. For the first time evidence has been obtained that filopodia, actin projections that radiate out from the surface of cells, serve as a route for the direct endocytosis of polyplexes. Confocal microscopy images demonstrated that filopodia on HeLa cells detect external polyplexes and extend into the extracellular milieu to internalize these particles. Polyplexes are observed to be internalized into membrane-bound vesicles (i.e., clathrin-coated pits and caveolae) directly within filopodial projections and are subsequently transported along actin to the main cell body for potential delivery of the nucleic acids to the nucleus. The kinetics and speed of polyplex trafficking have also been measured. The polyplex-loaded vesicles were also discovered to traffic between two cells within filopodial bridges. These findings provide novel insight into the early events of cellular contact with polyplexes through filopodial-based interactions in addition to endocytic vesicle trafficking-an important fundamental discovery to enable advancement of nonviral gene editing, nucleic acid therapies, and biomedical materials.