Folate receptors (FR) are cellular markers highly expressed in various cancer cells. Here, we report on the synthesis of a novel folate-containing lipoconjugate (FC) built of 1,2-di-O-ditetradecyl-rac-glycerol and folic acid connected via a PEG spacer, and the evaluation of the FC as a targeting component of liposomal formulations for nucleic acid (NA) delivery into FR expressing tumor cells. FR-targeting liposomes, based on polycationic lipid 1,26-bis(cholest-5-en-3β-yloxycarbonylamino)-7,11,16,20-tetraazahexacosan tetrahydrochloride (2X3), lipid helper dioleoylphosphatidylethanolamine (DOPE) and novel FC, formed small compact particles in solution with diameters of 60 ± 22 nm, and were not toxic to cells. Complexes of NAs with the liposomes were prepared at various nitrogen to phosphate ratios (N/P) to optimize liposome/cell interactions. We showed that FR-mediated delivery of different nucleic acids mediated by 2X3-DOPE/FC liposomes occurs in vitro at low N/P (1/1 and 2/1); under these conditions FC-containing liposomes display 3–4-fold higher transfection efficiency in comparison with conventional formulation. Lipoplexes formed at N/P 1/1 by targeted liposomes and cargo (Cy7-labeled siRNA targeting MDR1 mRNA) in vivo efficiently accumulate in tumor (˜15–18% of total amount), and kidneys (71%), and were retained there for more than 24 h, causing efficient downregulation of p-glycoprotein expression (to 40% of control) in tumors. Thus, FC containing liposomes provide effective targeted delivery of nucleic acids into tumor cells in vitro and in xenograft tumors in vivo.