A cholesterol-modified sphingomyelin (SM) lipid bilayer was developed to enhance therapeutic delivery. Cholesterol (Chol) improves lipid bilayer stability but is rapidly extracted under physiological conditions, leading to payload leakage. The study introduced SM-Chol, covalently conjugating Chol to SM, which retains Chol's membrane condensing ability. Systemic screening showed that SM-Chol with a disulfide bond and longer linker outperformed other systems in blocking Chol transfer and payload leakage, increasing the maximum tolerated dose of vincristine while reducing systemic toxicity. It improved pharmacokinetics, tumor delivery, and antitumor efficacy in lymphoma models. SM-Chol also enhanced delivery of various drugs and siRNA targeting P-glycoprotein in multiple cancer models. The bilayer's stability was enhanced by the amide bond in SM-Chol, which allows hydrogen bonding, and its double aliphatic chain increases bilayer compressibility. SM-Chol was more stable in physiological conditions and could be triggered to release its payload using stimuli-responsive bonds. The study demonstrated that SM-Chol significantly improved therapeutic delivery efficiency and safety, making it a promising platform for improved drug and gene delivery.A cholesterol-modified sphingomyelin (SM) lipid bilayer was developed to enhance therapeutic delivery. Cholesterol (Chol) improves lipid bilayer stability but is rapidly extracted under physiological conditions, leading to payload leakage. The study introduced SM-Chol, covalently conjugating Chol to SM, which retains Chol's membrane condensing ability. Systemic screening showed that SM-Chol with a disulfide bond and longer linker outperformed other systems in blocking Chol transfer and payload leakage, increasing the maximum tolerated dose of vincristine while reducing systemic toxicity. It improved pharmacokinetics, tumor delivery, and antitumor efficacy in lymphoma models. SM-Chol also enhanced delivery of various drugs and siRNA targeting P-glycoprotein in multiple cancer models. The bilayer's stability was enhanced by the amide bond in SM-Chol, which allows hydrogen bonding, and its double aliphatic chain increases bilayer compressibility. SM-Chol was more stable in physiological conditions and could be triggered to release its payload using stimuli-responsive bonds. The study demonstrated that SM-Chol significantly improved therapeutic delivery efficiency and safety, making it a promising platform for improved drug and gene delivery.