The present study aimed to evaluate the incorporation of the natural lipid lycopene into niosome formulations based on cationic lipid DOTMA and polysorbate 60 non-ionic surfactant to analyze the potential application of this novel formulation to deliver genetic material into the rat retina. Both niosomes with and without lycopene were prepared by the reverse phase evaporation method and physicochemically characterized in terms of size, zeta potential, polydispersity index and capacity to condense, release and protect the DNA against enzymatic digestion. In vitro experiments were performed in ARPE-19 cells after complexion of niosomes with pCMS-EGFP plasmid at appropriate cationic lipid/DNA ratios. At 18/1 mass ratio, nioplexes containing lycopene had nanometric size, positive zeta potential, low polydispersity and were able to condense, release and protect DNA. Percentage of transfected cell was around 35% without compromising cell viability. The internalization pathways studies revealed a preference to caveolae mediated endocytosis and macropinocytosis, which could circumvent lysosomal degradation. Both subretinal and intravitreal administrations to the rat retina showed that nioplexes were able to transfect efficiently the outer segments of the retina, which offer reasonable hope for the treatment of many inherited retinal diseases by a safe non-viral vector formulation after the less invasive intravitreal administration.