The potential, as non-toxic DNA vectors, of lipoplexes constituted by the DNA pEGFP-C3 (encoding the green fluorescent protein), a polycationic calixarene-based macrocyclic vector (CxCL) with a lipidic matrix (herein named TMAC4) and a zwitterionic lipid, the 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) has been analyzed from both biophysical and biochemical perspectives. For that purpose, several experimental methods, such as zeta potential (PALS methodology), agarose gel electrophoresis, small-angle X-ray scattering (SAXS), transmission electronic cryo-microscopy (cryo-TEM), atomic force microscopy (AFM), fluorescence microscopy, and cytotoxicity assays have been used. The electrochemical study shows that TMAC4 has 100% of its nominal charge available, while pDNA presents an effective negative charge that is only 10 % of its nominal one. PALS studies indicate the presence of three populations of nanoaggregates in TMAC4/DOPE lipid mixtures, with sizes around ∼ 100 nm, ∼ 17 nm and ∼ 6 nm, compatible with liposomes, oblate micelles, and spherical micelles, respectively, the first two being also detected by cryo-TEM. However, in the presence of pDNA, this mixture is organized in Lα multilamellar structures at all compositions. In fact, cryo-TEM micrographs show two types of multilamellar aggregation pattern: cluster-type (CT) at low and moderate CxCL molar fraction in the TMAC4/DOPE lipid mixture (α = 0.2 and 0.5), and fingerprint-type (FP), only present at low CxCL molar fraction (α = 0.2). This structural scenario has been also observed in SAXS diffractograms, including the coexistence of two different phases when DOPE dominates in the mixture. AFM experiments at α = 0.2 evidence that pDNA makes the lipid bilayer more deformable, thus provoking a potential enhance on the capability to penetrate the cells. In fact, the best transfection perfomances of these TMAC4/DOPE-pDNA lipoplexes have been obtained at low CxCL molar fraction (α = 0.2) and moderate-to-high effective charge ratio ρeff = 20. Probably, the coexistence of two lamellar phases is the responsible of the better TE performance at low α.