Although the transcription factor USF2 has been implicated in the regulation of cellular growth and proliferation, it is unknown whether alterations in USF2 contribute to tumorigenesis and tumor development. We examined the role of USF2 in prostate tumorigenesis. Western blot analysis revealed markedly decreased USF2 levels in three androgen-independent prostate cancer cell lines, PC-3, DU145, and M12, as compared to nontumorigenic prostate epithelial cells or the androgen-dependent cell line, LNCaP. Ectopic expression of USF2 in PC-3 cells did not affect the cell proliferation rate of PC-3 cells on plastic surfaces. However, it dramatically decreased anchorage-independent growth of PC-3 cells in soft agar (90-98% inhibition) and the invasion capability (80% inhibition) of PC-3 cells in matrix gel assay. Importantly, expression of USF2 in PC-3 cells inhibited the tumorigenicity of PC-3 cells in an in vivo nude mice xenograft model (80-90% inhibition). These results suggest that USF2 has tumor-suppression function. Consistent with its function in tumor suppression, we found that the USF2 protein is present in normal prostate epithelial cells but absent in 18 of 42 (43%) human prostate cancer tissues (P = 0.015). To further examine the functional role of USF2 in vivo, we generated mice with genetic deletion of USF2 gene. We found that USF2-null mice displayed marked prostate hyperplasia at a young age, suggesting that USF2 is involved in the normal growth and differentiation of prostate. Together, these studies demonstrate that USF2 has tumor-suppressor function and plays a role in prostate carcinogenesis.