Prostate cancer is the most prevalent malignancy and the second leading cause of cancer mortality in men. Early growth response gene-1 (EGR-1) plays a crucial role in the development and progression of prostate cancer. The presented data show that EGR-1 differs in cellular localization in benign cells compared with malignant prostate cells and that this localization is critical for the transcriptional activation of EGR-1-dependent genes. Immunohistochemistry of human prostate cancer specimens demonstrated higher levels of EGR-1 in malignant cells located predominantly in the cytoplasm, whereas benign cells contained lower levels of EGR-1 located predominantly in the nucleus. Benign prostate cells responded to mitogens in vitro, with increased levels of EGR-1, rapid nuclear translocation, and enhanced transcriptional activity, whereas malignant prostate cells did not exhibit the same responses, and the protein remained in the cytoplasm. The central aspect of this difference is the association of EGR-1 with microtubules, which is exclusive to the benign cells of the prostate and is requisite for the nuclear translocation and transcriptional activity of EGR-1. Our in vitro data demonstrate that the differences in EGR-1 between benign and malignant prostate cells extend beyond cellular levels, which was confirmed by immunohistochemistry in human tissues. Thus, we add the novel concept that microtubules regulate EGR-1 localization in benign prostate cells but not in malignant prostate cells.