An emerging class of natural surfactants, named alkylpolyglucosides, which can form both, the thermotropic and the lyotropic liquid crystalline phases, were focused. The aim of the study was to integrate some physicochemical properties (characterised through the polarization and transmission electron microscopy, wide-angle X-ray diffraction, thermal analysis and rheology) of the three formulations based on cetearyl glucoside and cetearyl alcohol, with the in vitro (the artificial skin constructs) and in vivo bioavailability of hydrocortisone (HC), in comparison with a standard pharmacopoeial vehicle. The parameters measured in vivo were erythema index (an instrumental human skin blanching assay), transepidermal water loss (TEWL) and stratum corneum hydration. A complex colloidal structure of lamellar liquid crystalline and lamellar gel crystalline type was deduced for sugar surfactant-based vehicles. In dependence on surfactant/water/oil ratio, several thermodynamically variable fractions of water were predicted. Rheological profile of the vehicle appeared to influence the in vitro profile of permeation. A surplus of total amount of drug permeated in vitro from the alkylpolyglucoside-based vehicles coincided with the more pronounced increase of TEWL and less marked blanching action of HC from the selected alkylpolyglucoside-based vehicle tested in vivo, related to the pharmacopoeial one. These findings imply an enhanced delivery of HC from this vehicle and its putative penetration enhancing effect, probably dependent on specific distribution of the vehicle's inherent water.