BACKGROUND AND OBJECTIVES: Arginase1 (ARG1) is an enzyme expressed by keratinocytes that drives several functions linked to skin barrier function. However, the mechanisms underpinning keratinocyte ARG1 function in barrier homeostasis is not fully elucidated. Atopic dermatitis (AD) is linked to impaired skin barrier via altered keratinocyte differentiation and susceptibility to infection. Therefore, we investigated the role of ARG1 in keratinocyte differentiation and anti-microbial responses. METHODS: In vitro 2D differentiation assays using ARG knockdown or ARG inhibited keratinocytes were used to explore the function of ARG1 in keratinocyte differentiation and barrier formation. ARG1 was also assessed in an ex vivo model of AD. RESULTS: ARG1 was strongly expressed in the apical layers of human skin, corresponding with high ARG1 expression in late differentiated keratinocytes. ARG was downregulated in an ex vivo AD model relative to controls, suggesting altered ARG1 is clinically relevant. ARG1 inhibition in keratinocytes led to a significant decrease in late differentiation markers Filaggrin (FLG), Involucrin (IVL), and Loricrin (LOR) and significant downregulation of the Anti-microbial Peptides (AMPs), Lipocalin 2 (LCN2), Kallikreins (KLKs) and Small Proline Rich Proteins (SPRRs). ARG forms part of the urea cycle and the action of ARG on L- arginine causes the production of L-ornithine and urea. L-ornithine, in turn, is catabolised for putrescine (Put) production. Supplementation with ARG products, Put and urea, could rescue late keratinocyte differentiation and AMP expression in ARG deficient cells. CONCLUSIONS: ARG1 activity plays a major role in keratinocyte differentiation and AMP production. ARG1 is downregulated in AD, but in cell systems is amenable to rescue by ARG1 downstream products Put and urea. Manipulation of the ARG1 pathway may, therefore, have potential to be used for the management of skin conditions such as AD.