Non-viral gene carriers have been extensively investigated as alternatives to viral vectors for gene delivery systems into animal and plant cells. A non-viral gene carrier containing a cell-penetrating peptide and a cationic sequence was previously developed for use in intact plants and plant cells; however, the permeation pathway of the gene carrier into plant cells is yet to be elucidated, which would facilitate the improvement of the gene delivery efficiency. Here, we identified the vacuum/compression infiltration-mediated permeation pathway of a non-viral gene carrier into plant tissues and cells using a complex of plasmid DNA and a peptide-based gene carrier. This complex was taken up via the hydathodes in Arabidopsis thaliana, and from root hairs in Nicotiana benthamiana. Remarkably, these structurally weak tissues are also routes of bacterial invasion in nature, suggesting that peptide-pDNA complexes invade intact plants through similar pathways as bacterial pathogens.