The study introduces a cholesterol-modified sphingomyelin (SM) lipid bilayer, named SM-Chol, which retains the membrane condensing ability of cholesterol while preventing its rapid extraction from the bilayer under physiological conditions. Systematic structure-activity relationship (SAR) screening reveals that SM-Chol with a disulfide bond and a longer linker outperforms other systems in blocking cholesterol transfer and payload leakage, increasing the maximum tolerated dose of vincristine, improving pharmacokinetics and tumor delivery efficiency, and enhancing antitumor efficacy in a diffuse large B-cell lymphoma xenograft model. SM-Chol also improves the therapeutic delivery of structurally diverse drugs (irinotecan, doxorubicin, dexamethasone) and siRNA targeting the multi-drug resistant gene (p-glycoprotein) in various cancer and inflammatory disease models compared to FDA-approved nanotherapeutics or lipid compositions. These findings suggest that SM-Chol represents a promising platform for improved drug and gene delivery.The study introduces a cholesterol-modified sphingomyelin (SM) lipid bilayer, named SM-Chol, which retains the membrane condensing ability of cholesterol while preventing its rapid extraction from the bilayer under physiological conditions. Systematic structure-activity relationship (SAR) screening reveals that SM-Chol with a disulfide bond and a longer linker outperforms other systems in blocking cholesterol transfer and payload leakage, increasing the maximum tolerated dose of vincristine, improving pharmacokinetics and tumor delivery efficiency, and enhancing antitumor efficacy in a diffuse large B-cell lymphoma xenograft model. SM-Chol also improves the therapeutic delivery of structurally diverse drugs (irinotecan, doxorubicin, dexamethasone) and siRNA targeting the multi-drug resistant gene (p-glycoprotein) in various cancer and inflammatory disease models compared to FDA-approved nanotherapeutics or lipid compositions. These findings suggest that SM-Chol represents a promising platform for improved drug and gene delivery.