Summary Background The epidermal barrier is important for water conservation, failure of which is evident in dry‐skin conditions. Barrier function is fulfilled by the stratum corneum, tight junctions (TJs, which control extracellular water) and keratinocyte mechanisms, such as organic osmolyte transport, which regulate intracellular water homeostasis. Organic osmolyte transport by keratinocytes is largely unexplored and nothing is known regarding how cellular and extracellular mechanisms of water conservation may interact. Objectives We aimed to characterize osmolyte transporters in skin and keratinocytes, and, using transporter inhibitors, to investigate whether osmolytes can modify TJs. Such modification would suggest a possible link between intracellular and extracellular mechanisms of water regulation in skin. Methods Immunostaining and quantitative polymerase chain reaction of organic osmolyte‐treated organ‐cultured skin were used to identify changes to organic osmolyte transporters, and TJ protein and gene expression. TJ functional assays were performed on organic osmolyte‐treated primary human keratinocytes in culture. Results Immunostaining demonstrated the expression of transporters for betaine, taurine and myo‐inositol in transporter‐specific patterns. Treatment of human skin with either betaine or taurine increased the expression of claudin‐1, claudin‐4 and occludin. Osmolyte transporter inhibition abolished this response. Betaine and taurine increased TJ function in primary human keratinocytes in vitro. Conclusions Treatment of skin with organic osmolytes modulates TJ structure and function, which could contribute to the epidermal barrier. This emphasizes a role for organic osmolytes beyond the maintenance of intracellular osmolarity. This could be harnessed to enhance topical therapies for diseases characterized by skin barrier dysfunction. What is already known about this topic? Limited studies have shown that organic osmolytes protect keratinocytes from stressors such as ultraviolet, but their transporters have never been shown in human skin. What does this study add? Human skin expresses the transporters for betaine, taurine and myo‐inositol and their expression is increased by the respective osmolytes. We show that osmolytes increase tight junction protein expression in skin and tight junction function in keratinocytes by a variety of mechanisms. What is the translational message? Osmolytes can act as signalling molecules, which change essential components of the barrier. This suggests that their inclusion in topical therapies might help improve the epidermal barrier in healthy skin and also in diseases where the barrier is dysfunctional. Linked Comment: Benedetto. Br J Dermatol 2021; 184:388–389. Plain language summary available online