Abstract: Glucocorticoids, crucial regulatory hormones involved in the stress response, significantly influence growth, development, and metabolism through activation of the glucocorticoid receptor (GR). Hypoxia-inducible factor 3 alpha (HIF-3α), the least characterized paralog among three HIF-α proteins, plays a role in adaptation to oxygen level changes and metabolic reprogramming. Despite the potential functional overlaps between GR and HIF-3α pathways in regulating metabolism, their crosstalk remains poorly understood. Here, we demonstrate a regulatory mechanism governing the crosstalk between these two transcription factor pathways. We found that upon ligand activation, GR binds to the intronic region of the HIF3A gene and upregulates its mRNA transcription. Additionally, HIF-3α and GR engage in protein–protein interactions through the oxygen-dependent degradation domain of HIF-3α and all major domains of GR (i.e. the N-terminal, DNA-binding, and ligand-binding domains). Furthermore, we discovered that this interaction results in reciprocal attenuation of the transcriptional activities of both GR and HIF-3α, causing a negative feedback loop upon HIF3A gene expression. The GR-HIF-3α interaction may offer a targetable pivot to modulate these two TF pathways, potentially providing a novel therapeutic avenue for related diseases.