Non-viral gene delivery systems have proven to be a promising approach in the treatment of brain metastatic cancers but facing delivery difficulties. Due to the existence of blood-brain barrier, non-viral gene carriers must pass through brain capillary endothelial cells to accumulate at the brain tumor sites. However, during this process, most of them trap into brain capillary endothelial cells and fail to penetrate to the brain tumor sites. Autophagy is involved in dynamic disposition of both intracellular and extracellular components, which theoretically affects intracellular fate of non-viral gene carriers during BBB penetration. In the present study, R6dGR peptide-modified PEGylated polyethyleneimine that carry therapeutic gene encoding human tumor necrosis factor-related apoptosis-inducing ligand (PPR/pTRAIL) are established as model non-viral gene delivery system and applied in breast cancer brain metastasis therapy. Autophagy-mediated lysosome degradation pathway is found to be involved in the degradation of PPR/pTRAIL in brain capillary endothelial cells and prevents them from BBB penetration. Pre-inhibiting BBB autophagy level by wortmannin loaded liposomes (Wtmn-Lip) can increase brain accumulation of non-viral gene carrier PPR without damaging BBB tight junctions. Besides, Wtmn-Lip synergistically induces apoptosis with TRAIL via different signaling pathways. Herein, pre-treatment of Wtmn-Lip might solve delivery difficulties of non-viral gene carriers in the treatment of brain metastatic cancers.