Circadian (~ 24 h) control impinges on an array of diverse physiological processes in many organisms, ranging from plants to human. Disruption of the mammalian circadian clockwork can lead to severe chronic illnesses such as cardiovascular disease, cancer progression and metabolic disorders. Transcriptional regulation of plasminogen activator inhibitor 1 (PAI-1) is of particular importance because of its crucial role in these pathological conditions. Pai-1 expression is partly regulated by the circadian clock, although direct mechanisms on Pai-1 rhythmicity are unknown. In the present study, we have identified a conserved functional E-box cis-element in the distal part of the mouse Pai-1 gene that is necessary and sufficient to drive circadian expression in Pai-1 activity after dexamethasone synchronisation in vitro. Mutagenesis and in vitro transfection analysis indicated this E-box provides a cognate binding site for cross-talk between clock and hypoxia factors, thus providing a potential cooperation mechanism between circadian and stress pathways, which is conserved in the human Pai-1 gene. Together, these results suggest that the canonical E-box is a target for glucocorticoid action, thus providing the molecular interface between gene transcription and drug action. The mechanism described has global impact on diverse dynamic biological processes governed by the neuroendocrine axis and the circadian clockwork to control complex coordination of gene cascades and biology.