The insulin-like peptide relaxin is a central hormone of pregnancy, but it also produces antifibrotic, myocardial, renal, central nervous, and vascular effects. Recently, two G-protein-coupled receptors, LGR7 and LGR8, were identified as relaxin receptors. Prompted by reports on the immunoregulatory effects of relaxin, we investigated possible interactions with the human glucocorticoid receptor (GR). Relaxin blunted the endotoxin-induced production of inflammatory cytokines (interleukin 1 [IL-1], IL-6, and tumor necrosis factor- alpha) by human macrophages, an effect that was suppressed by the GR antagonist RU-486. In three different cell lines, relaxin induced GR activation, nuclear translocation, and DNA binding as assessed in glucocorticoid response element (GRE)-luciferase assays. Coimmunoprecipitation experiments revealed physical interaction of endogenous and exogenous relaxin with cytoplasmic and nuclear GR. Relaxin competed with GR agonists for GR binding both in vivo, in whole-cell assays, and in vitro, in fluorescence polarization assays. In LGR7- and LGR8-free cells, the relaxin-mediated activation of GR was preserved. In conclusion, relaxin acts as a GR agonist, a pathway pivotal to relaxin's effects on cytokine secretion by human macrophages. These findings may deepen our understanding of relaxin's many physiologic actions as well as our insights into general principles of hormone signaling.