Significant effort continues to be exerted toward the improvement of transfection mediated by nonviral vectors. These endeavors are often focused on the design of particulate carriers with properties that encourage efficient accumulation at the membrane surface, particle uptake, and endosomal escape. Despite its demonstrated importance in successful nonviral transfection, relatively little investigation has been done to understand the pressures driving internalized vectors into favorable nondegradative endocytic pathways. Improvements in transfection efficiency have been noted for complexes delivered with a substrate-mediated approach, but the reasons behind such enhancements remain unclear. The phenotypic changes exhibited by cells interacting with nano- and micro-featured substrates offer hints that may explain these effects. This review describes nanoscale particulate and substrate parameters that influence both the uptake of nonviral gene carriers and the endocytic phenotype of interacting cells, and explores the molecular links that may mediate these interactions. Substrate-mediated control of endocytosis represents an exciting new design parameter that will guide the creation of efficient transgene carriers.