Folate receptors are overexpressed on the surface cancer cells. We successfully constructed a new gene delivery vector of methotrexate (MTX)-coated plasmid DNA–polyethylenimine (pDNA–PEI) complexes (PEI complexes) by electrostatic binding. The stable anionic nanoparticle was optimized at MTX charge ratios of 120 or more. pDNA–PEI–MTX complexes (MTX complexes) demonstrated gene expression efficiency as high as cationic pDNA–PEI complexes in the mouse melanoma cell line, B16-F10. The MTX complexes were taken up by the cell-specific uptake mechanisms via the folate receptor. MTX-coated complexes are useful as endocytosis ligands. The MTX120 complexes exhibited no blood aggregation. The transgene efficiency of MTX120 complexes in the liver and spleen after their intravenous administration was higher than that of PEI complexes. Therefore, MTX complexes are expected as a new gene vector in the future.