Extensive research is currently performed on designing safe and efficient non-viral carriers for gene delivery. To increase their efficiency, it is essential to have a thorough understanding of the mechanisms involved in cellular attachment, internalization and intracellular processing in target cells. In this work, we studied in vitro the cellular dynamics of polyplexes, composed of a newly developed bioreducible poly(amido amine) carrier, formed by polyaddition of N,N-cystamine bisacrylamide and 1-amino-4-butanol (p(CBA-ABOL)) on retinal pigment epithelium (RPE) cells, which are attractive targets for ocular gene therapy. We show that these net cationic p(CBA-ABOL)/DNA polyplexes require a charge-mediated attachment to the sulfate groups of cell surface heparan sulfate proteoglycans in order to be efficiently internalized. Secondly, we assessed the involvement of defined endocytic pathways in the internalization of the polyplexes in ARPE-19 cells by using a combination of endocytic inhibitors, RNAi depletion of endocytic proteins and live cell fluorescence colocalization microscopy. We found that the p(CBA-ABOL) polyplexes enter RPE cells both via flotillin-dependent endocytosis and a PAK1 dependent phagocytosis-like mechanism. The capacity of polyplexes to transfect cells was, however, primarily dependent on a flotillin-1-dependent endocytosis pathway.