The endocrine system participates in regulating macrophage maturation, although little is known about the modulating role of the thyroid hormones. In vitro results demonstrate a negative role of one such hormone, triiodothyronine (T₃), in triggering the differentiation of bone marrow–derived monocytes into unpolarized macrophages. T₃-induced macrophages displayed a classically activated (M1) signature. A T₃-induced M1-priming effect was also observed on polarized macrophages because T₃ reverses alternatively activated (M2) activation, whereas it enhances that of M1 cells. In vivo, circulating T₃ increased the content of the resident macrophages in the peritoneal cavity, whereas it reduced the content of the recruited monocyte-derived cells. Of interest, T₃ significantly protected mice against endotoxemia induced by lipopolysaccharide i.p. injection; in these damaged animals, decreased T₃ levels increased the recruited (potentially damaging) cells, whereas restoring T₃ levels decreased recruited and increased resident (potentially beneficial) cells. These data suggest that the anti-inflammatory effect of T₃ is coupled to the modulation of peritoneal macrophage content, in a context not fully explained by the M1/M2 framework. Thyroid hormone receptor expression analysis and the use of different thyroid hormone receptor antagonists suggest thyroid hormone receptor β1 as the major player mediating T₃ effects on macrophages. The novel homeostatic link between thyroid hormones and the pathophysiological role of macrophages opens new perspectives on the interactions between the endocrine and immune systems.