The skin barrier provides protection from external threats, and defects in the skin barrier are involved in several skin diseases such as atopic dermatitis, eczema, and psoriasis. Growing evidence suggests that endocannabinoids (EC) have a role in the biological processes of the skin including skin barrier formation. The EC system involvement in other biological barriers such as intestinal barrier, bronchial barrier and blood brain barrier has been shown to involve regulation of the tight junction (TJ) formation; this leads to the hypothesis that the EC system may also be involved in skin barrier regulation through regulation of epidermal TJ. The aim of this study was to explore the role of EC in keratinocyte differentiation and TJ functionality, two important processes in skin barrier formation, and explore the modulation of the EC system by the anti-inflammatory omega-3 (n-3) polyunsaturated fatty acids (PUFA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). In order to address this aim, HaCaT keratinocytes, primary normal human epidermal keratinocytes (NHEK) and human skin samples were used as models. UVR and histamine were used to disrupt the barrier. Healthy volunteers were given EPA and DHA as oral supplementation and donated UVR-treated skin. EC and related N-acyl ethanolamines (NAE) and monoacyl glycerols (MAG) were analysed by UPLC/ESI-MS/MS in cells and skin samples. Enzymes (NAPE-PLD, 12-LOX, and 15-LOX2) and Langerhans cells (LC) were analysed by IHC. TJ functionality was assessed by transepithelial electrical resistance (TEER) and FITC-dextran permeability assays. Localisation of TJ proteins was assessed by IF, and protein and gene expression by western blot and RT-qPCR, respectively. EC and NAE were detected in cells and human skin samples. N-palmitoyl ethanolamine (PEA) was effective on TJ and two different paracellular pathways were investigated: the pore pathway responsible for the passage of small ions and leak pathway responsible for the passage of large molecules. TEER and FITC-Dextran assays revealed that PEA increased TJ integrity (p=0.0419) but had no effect on permeability in NHEK. PEA appears to mediate the pore pathway, supported by increased expression of claudin-4 protein (p=0.020) and claudin-4 gene expression (p=0.0011). PEA did not affect the expression of occludin and zonula occluden-1, responsible for the leak pathway. UVR did not alter the production of EC and NAE. However, increases in PEA (p=0.035), N-oleoyl ethanolamine (OEA) (p=0.022) and N-vaccenoyl ethanolamine (VEA) (p=0.024) levels in primary NHEK were observed overtime in response to histamine; this shows that NAE are involved in histamine-induced skin barrier damage. Interestingly, PEA (p=0.0123) was also able to repair the damaging effect of histamine on TJ, as shown by increased TEER. EPA and DHA had differential effects on EC in UVR-challenged skin. DHA overall decreased EC production and NAPE-PLD expression, while EPA showed an opposite effect and increased 15-LOX2. Only DHA reduced the migration of LC caused by UVR, while EPA reduced the effect of UVR on epidermal thickness. Overall, this study showed that EC and NAE are involved in skin barrier formation through TJ; PEA was the most potent species affecting TJ functionality acting through regulation of the TJ pore pathway. Production of EC and NAE are differentially regulated by EPA and DHA, and DHA could be used to modulate the EC system. Altering skin EC may lead to a development of potential barrier therapies by improving the integrity of epidermal TJ.