15-lipoxygenase 2 (15-LOX-2) is lost or significantly reduced in prostate cancer. However, the regulation of 15-LOX-2 remains unclear. In this study, we independently cloned the 5' upstream promoter fragments of 15-LOX-2 gene. Target DNA fragments each were cloned into an expression vector containing luciferase reporter gene, which were called LF1(-1533/+87), LF2(-628/+87), LF3(-253/+87), LF4(-157/+87), LF5(-33/+87), LF6(-253/+1), and LF7(-157/+1). Each of these individual promoter fragments was transfected into primary prostate epithelial cells and prostate cancer LNCaP cells. The promoter activity gradually decreased with progressive deletions from LF2 to LF4. A significant drop was noted in the LF5. LF6 and LF7 that did not contain the 87-bp region downstream the transcription start site (TSS) have significant luciferase activities similar to those of corresponding fragments (LF3 and LF4) that contain 87-bp region downstream the TSS. This suggests that the 125-bp region (-157 to -33) of LF4 is critical for the promoter activity of 15-LOX-2 in the primary prostate epithelial cells PrEC and cancer cells LNCaP. Moreover, we discovered a specific glucocorticoid receptor (GR) responsive element (GRE) in this key region. The luciferase activities of the LF4 and LF7 were decreased in the LNCaP cells co-transfected with GR (hGRalpha or hGRbeta) expression vectors. This inhibitory effect is reversed after treatments with dexamethasone or two specific GR inhibitors (siRNAs of GR and RU486). Results from this study suggest a 125-bp region (-157 to -33) is critical for the 15-LOX-2 promoter activity in prostate epithelial cells and cancer cells, which was significantly downregulated by GR via the GRE in this region.