Epidermal barrier function is provided by the highly keratinised stratum corneum and also by tight junctions (TJs) in the granular layer of skin. The development of the TJ barrier significantly deteriorates in response to ultraviolet B radiation (UVB). Following exposure to UVB, keratinocytes accumulate organic osmolytes, which are known to preserve cell volume during water stress. Since TJs are intimately associated with control of water homeostasis in skin, we hypothesised that there may be a direct influence of osmolytes on TJ development. Exposure of rat epidermal keratinocytes (REKs) to a single dose of UVB reduced the function of developing TJs. This was concomitant with dislocalisation of claudin-1 and claudin-4 from the keratinocyte plasma membrane, phosphorylation of occludin and elevation of reactive oxygen species (ROS). In the presence of organic osmolytes, these effects were negated but were independent of the effects of these molecules on cell volume, elevation of ROS or the gene expression of TJ proteins. These data suggest that organic osmolytes affect TJs via post-translational mechanism(s) possibly involving protection of the native conformation of TJ proteins.