A simple and economic methodology to synthesize three types of novel dimeric cholesterol derivatives (DCDs) was developed. Results obtained from dynamic light scattering and transmission electron microscopy show that spherical and/or angular nano-structural aggregates of DCDs are formed by self-assembly in aqueous solution. The size and morphology of DCD dispersions depend on the spatial arrangement of the substituents and polarity of the head group in the DCD structures. The cytoxicity of DCD dispersions to human keratinocytes (HaCaT) and squamous cell carcinomas (SCC25) cells was also evaluated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The present novel DCD dispersions were not toxic to HaCaT and SCC25 cells at appropriate tested concentrations. Crown Copyright (C) 2013 Published by Elsevier B.V. All rights reserved.