The glucocorticoid receptor (GR) can bind to DNA or RNA, eliciting transcriptional activation/repression or rapid messenger RNA (mRNA) degradation, respectively. Although GR-mediated transcriptional regulation has been well-characterized, the molecular details of rapid mRNA degradation induced by glucocorticoids are not yet fully understood. Here, we demonstrate that glucocorticoid-induced GR-mediated mRNA decay (GMD) takes place in the nucleus and the cytoplasm, acting on pre-mRNAs and mRNAs. We also performed cross-linking and immunoprecipitation coupled with high-throughput sequencing analysis for GMD factors (GR, YBX1, and HRSP12) and mRNA sequencing analysis to identify endogenous GMD substrates. Our comprehensive coupled with high-throughput sequencing and mRNA sequencing analyses reveal that a range of cellular transcripts containing a common binding site for GR, YBX1, and HRSP12 are preferential targets for GMD, suggesting possible new functions of GMD in various biological events.