N‑Acylserinols (NASOHs) exhibit anti-cancer activity by elevating ceramide levels, and/or by activating proapoptotic effectors. In the present work we investigated the thermotropic phase behavior and supramolecular organization of a homologous series of NASOHs (number of C-atoms in the acyl chain, n = 8-18), and the interaction of N-myristoylserinol (NMSOH) with cholesterol, and characterized cationic niosomes made up of NMSOH, cholesterol and cetyltrimethylammonium bromide (CTAB). Differential scanning calorimetric studies revealed that NASOHs exhibit a major chain-melting phase transition in both dry and hydrated states. The thermodynamic parameters, transition enthalpy and entropy show linear dependence on the acyl chain length in the dry state, but exhibit odd-even alternation in the hydrated state. Powder X-ray diffraction studies revealed that NASOHs adopt a tilted bilayer structure, wherein the bilayer repeat distances (d-spacings) also showed odd-even alteration, with even-chainlength compounds exhibiting slightly higher d-spacings. Studies on the interaction between NMSOH and cholesterol revealed that both lipids mix well with up to 55 mol% cholesterol, whereas phase separation was observed at higher cholesterol content. The transition enthalpy corresponding to the NMSOH-cholesterol complex increases up to 55 mol% cholesterol and decreases at higher cholesterol content. Presence of the cationic surfactant CTAB affects the phase behavior, fluidity and size of the NMSOH-cholesterol (45,55, mol/mol) niosomes, with unilamellar vesicles of about 85 (±20) nm in diameter being obtained at 10 mol% CTAB. These results provide a thermodynamic and structural basis for further investigations on these cationic niosomes towards their use in drug delivery applications, especially for anticancer drugs. Copyright © 2018 Elsevier B.V. All rights reserved.