Androgen ablation therapy is an effective treatment for advanced prostate cancer, but the tumor often progresses toward a more aggressive phenotype. We determined the changes in genes associated with the malignant progression and found increased thymosin beta4, involved in tumor metastasis, in androgen-sensitive LNCaP cells grown in the medium with androgen-deficient, charcoal-stripped fetal calf serum. The mRNA expression of thymosin beta4 was determined by real-time polymerase chain reaction analysis. The transcriptional activity of thymosin beta4 was measured by luciferase assay using reporter plasmid containing 5'-flanking region of thymosin beta4. Thymosin beta4 mRNA expression was increased in LNCaP cells in the androgen-deficient condition and decreased by dihydrotestosterone treatment. Androgen receptor antagonist bicalutamide inhibited thymosin beta4 expression in a dose-dependent manner. In androgen receptor-negative PC-3 cells, no significant effects on thymosin beta4 gene expression were observed. The regulation of thymosin beta4 mRNA expression by androgen is due to the transcriptional activation. Deletion analysis revealed that the region between -83 bp and -46 bp of the thymosin beta4 gene is responsible for the regulation of the transcriptional activity by androgen. Thymosin beta4 expression is negatively controlled at the transcriptional level by androgen.