Significance Glucocorticoids (GCs), acting through binding to the GC receptor (GR), are peripheral effectors of circadian and stress-related homeostatic functions fundamental for survival throughout vertebrate life span. They are widely used to combat inflammatory and allergic disorders, and their therapeutic effects have been mainly ascribed to their capacity to suppress the production of proinflammatory cytokines. The present study unveils, at the molecular level, the mechanisms that underlie the GC-induced GR direct transrepression function mediated by the evolutionary conserved inverted repeated negative response element. This knowledge paves the way to the elucidation of the functions of the GR at the submolecular levels and to the future educated design and screening of drugs, which could be devoid of undesirable debilitating effects on prolonged GC therapy.