Metabolic reprogramming of macrophages initiates the polarization of pro-inflammatory macrophages that exacerbates adipocyte dysfunction and obesity. The imbalance of mitochondrial Ca homeostasis impairs mitochondrial function and promotes inflammation. Connexin 43 (Cx43), a ubiquitous gap junction protein, has been demonstrated to regulate intracellular Ca homeostasis. Here we explored whether macrophage Cx43 affects the obesity process by regulating the polarization of macrophage. HFD treatment induced obesity and exacerbated macrophages infiltration with upregulation of macrophages Cx43. Macrophage-specific knockout of Cx43 reduced HFD-induced obesity by alleviating inflammation in adipose tissue, with less pro-inflammatory M1 macrophage infiltration. Consistently, inhibition or knockdown of Cx43 improved palmitic acid (PA) induced mitochondrial dysfunction, as indicated by improved oxidative phosphorylation (OXPHOS), reduced formation of mitochondria-associated membranes (MAM) and mitochondrial Ca overload. Mechanistically, Cx43 interacted with the mitochondrial Ca uniporter (MCU) and knockdown of Cx43 alleviated PA-induced succinate dehydrogenase (SDH) oxidation by lowering MCU-mediated mitochondrial Ca uptake, which then, promoting the polarization of pro-inflammatory M1 macrophages. Thus, this study identified Cx43 as a mitochondrial Ca regulator that aggravates obesity via promoting macrophages polarized to M1 pro-inflammatory phenotype and suggests that Cx43 might be a promising therapeutic target antagonizing obesity.