Pathologically impaired healing in the elderly remains a major area of unmet clinical need that imposes a significant financial burden on the world's health services, along with severe patient morbidity. In women the transition to delayed healing coincides with menopause, where hormone levels, particularly estrogen, rapidly fall. Our group have previously shown that estrogen, rather than intrinsic ageing, is a major regulator of delayed healing in the elderly and that estrogen replacement, in both human and murine models, significantly improves healing. The aim of this study has been to functionally explore the role of estrogen signalling during healing, with a particular focus on the inflammatory response. Using transgenic mouse models, we present compelling new data that reveals diverse roles for the two estrogen receptors (ER) during healing. Specifically, in vivo cre-mediated ER ablation studies provide clear evidence that epidermal ERβ and inflammatory-cell ERalpha are important to mediate estrogen's beneficial effects on healing. Subsequent microarray analysis, of global ER null mice confirm a key role for ERβ mediating epidermal gene expression while ERalpha is required to regulate inflammatory-cell mediators. This led us to focus on the role of macrophage polarisation during healing, where we present data demonstrating dysregulation of macrophage polarisation in pre-clinical delayed healing models. Collectively, our in vivo and in vitro studies show that estrogen is able to promote alternative macrophage activation, mediated via ERalpha. Finally, we use a combined pharmacological inhibition and conditional genetic deletion approach to demonstrate a key role for the alternatively activated macrophage marker arginase during healing in both mouse and human. Overall, this research has significantly improved our understanding of the hormonal regulation of healing and highlighted important therapeutic targets that may be employed for the treatment of both acute and chronic wounds.