Glucocorticoids regulate diverse physiological functions ranging from mitosis to apoptosis, although only one glucocorticoid receptor (GR) gene has been discovered. We report here that one single GR mRNA species unexpectedly produces at least eight functional GR N-terminal isoforms via translational mechanisms. These GR isoforms display diverse cytoplasm-to-nucleus trafficking patterns and distinct transcriptional activities. In human osteosarcoma cells, the transcriptional responses to glucocorticoids closely reflect the identity and abundance of the GR isoforms. In addition, each GR isoform regulates both a common and a unique set of genes in the same cell. Interestingly, the levels of these GR isoforms differ significantly among tissues. Based on these observations, we propose that cell-type specific GR isoforms generate specificity in glucocorticoid control of transcription in different tissues.