Reductions in estrogen receptor (ER) expression are associated with metabolic dysfunction and heightened disease risk in women and men. However, the causal mechanisms underlying reduced ERα levels in the context of metabolic dysfunction and the specific tissue(s) conferring ERα-mediated effects on metabolism, inflammation, and insulin action remain inadequately defined. We have shown that aspects of the metabolic syndrome including insulin resistance, tissue inflammation, and obesity are recapitulated in whole body Esr1-/- mice (Esr1 encodes ERα). We have subsequently performed a tissue dissection approach to understand the cell-specific impact of ERα action on inflammation and metabolic homeostasis. Because macrophages (MΦ) are a key cell type regulating tissue metabolism, and are involved in the pathobiology of cardiometabolic-related diseases, herein we propose to interrogate the mechanisms by which ERα modulates MΦ function with a specific focus on M mitochondrial metabolism and mtDNA replication. In Aim 1 we will use loss- (MACER) and gain-of-Esr1 (ERαMyeTg) expression approaches to selectively modulate ERα in the myeloid lineage of male and female mice. We will examine the impact of ERα expression on metabolic homeostasis in response to high fat diet feeding and determine whether restoration of ERα in myeloid cells of the whole body Esr1-/- mouse model can reverse the obesity and insulin resistance phenotype. Since we were the first to identify that ER regulates mitochondrial dynamics and mtDNA replication in myocytes and adipocytes, and since mitochondria are viewed as central signaling hubs regulating immunometabolism, in Aim 2 we will interrogate the role of ERα in controlling fission/fusion/mitophagy dynamics and mtDNA replication by polymerase γ, Polg1 (catalytic subunit). Since our research shows contrasting molecular outcomes to ERα deletion in different cell types, a primary goal of this proposal is to better understand the role of ERα in controlling mitochondrial function specifically in MΦ, as well as determine how impaired ERα action drives inflammation and metabolic dysfunction with relevance to cardiometabolic disease susceptibility, especially in women during the menopausal transition.