Glucocorticoid hormones (GC) regulate essential physiological functions including energy homeostasis, embryonic and postembryonic development, and stress response. From the biomedical perspective, GC have garnered a tremendous amount of attention as highly potent anti-inflammatory and immunosuppressive medications indispensable in the clinic.GC signal through the GC receptor (GR), a ligand-dependent transcription factor whose structure, DNA binding, and the molecular partners that it employs to regulate transcription have been under intense investigation for decades. In particular, next-generation sequencing-based approaches have revolutionized the field by introducing a unified platform for a simultaneous genome-wide analysis of cellular activities at the level of RNA production, binding of transcription factors to DNA and RNA, and chromatin landscape and topology. Here we describe fundamental concepts of GC/GR function as established through traditional molecular and in vivo approaches and focus on the novel insights of GC biology that have emerged over the last 10 years from the rapidly expanding arsenal of system-wide genomic methodologies. Expected final online publication date for the Annual Review of Physiology Volume 78 is February 10, 2016. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.