To confirm whether autophagy regulation plays a synergistic role in enhancing the antitumor efficacy of a chemotherapeutic drug, in this work, we prepared chitosan (CS) NPs with the combined co-delivery of gefitinib and short hairpin (sh)RNA-expressing plasmid DNA targeting the Atg-5 gene (shAtg-5) to improve anti-cancer effects. The gene discussed is ATG5; the disease is cancer.