Context: Transdermal formulations contain permeation enhancer which causes skin damage. Ceramide 2 is natural lipid found in stratum corneum (SC).Objective: Drug-loaded nanovesicles of ceramide-2, cholesterol, palmitic acid, cholesteryl sulfate were formulated and analyzed for physical and biological properties. Diclofenac was used as a model drug.Materials and method: The vesicles were prepared using the film hydration method and characterized for physical parameters, in vitro drug release, accelerated stability studies and formulated into gel. Respective gels were compared with a commercial formulation (CEG) and plain carbopol gel (CG) containing drug for ex vivo, in vivo drug permeation and anti-inflammatory activity.Results: The vesicles were stable with optimum physical parameters. DCG-1 showed 92.89% in vitro drug release. Ceramide vesicles showed drug release between 18 and 25 μg/cm2 whereas CG and CEG released 0.33 and 1.35 μg/cm2 drug, respectively. DCG-1 and CEG showed corresponding Cmax at 6 and 4 h, respectively. DCG-1 showed six times AUC than CEG. DCG-1 inhibited edema by 86.37% by 4th hour of application.Discussion: The presence of ceramide 2 specifically promotes the drug permeation through SC and dermis and also contribute towards stability and non-irritancy.Conclusion: The composition of the nanovesicle played an important role in physical properties and drug permeation.