Glucocorticoids regulate a variety of physiological processes and are commonly used to treat disorders of inflammation, autoimmune diseases, and cancer. Glucocorticoid action is predominantly mediated through the classic glucocorticoid receptor (GR)α isoform. Recent data suggest that the mature GRα mRNA is translated into multiple N-terminal isoforms that have distinct biochemical properties and gene regulatory profiles. Interestingly, osteosarcoma cells stably expressing the GRα-D translational isoform are unique in that they are resistant to glucocorticoid-induced apoptosis. In this study, we investigate whether GRα isoform-specific differences in the regulation of antiapoptotic genes contribute to this resistant phenotype. We now show that GRα-D, unlike the other receptor isoforms, does not inhibit the activity of a nuclear factor κB (NF-κB)-responsive reporter gene and does not efficiently repress either the transcription or protein production of the antiapoptotic genes Bcl-xL, cellular inhibitor of apoptosis protein 1, and survivin. The inability of GRα-D to down-regulate the expression of these genes appears to be associated with a diminished interaction between GRα-D and NF-κB that is observed in cells, but not in vitro, and likely reflects the sequestration of GRα-D in the nucleus. Deletion of the GRα N-terminal amino acids 98-335 also results in a nuclear resident GR, which fails to interact with NF-κB in cells and promote apoptosis in response to glucocorticoids. These data suggest that the N-terminal translational isoforms of GRα selectively regulate antiapoptotic genes and that the GRα-D isoform may contribute to the resistance of certain cancer cells to glucocorticoid-induced apoptosis.