We have recently found that analog V (BXL-353, a calcitriol analog), inhibits growth factor (GF)-stimulated human benign prostate hyperplasia (BPH) cell proliferation by disrupting signal transduction, reducing Bcl-2 expression and inducing apoptosis. We now report that BXL-353 blocks in vitro and in vivo testosterone (T) activity. BPH cells responded to T and to dihydrotestosterone (DHT) with a dose-dependent growth and reduced apoptosis. Exposure of BPH cells to BXL-353 significantly antagonized both T- and DHT-induced proliferation, and induced apoptosis, even in the presence of T. To verify whether BXL-353 reduced prostate growth in vivo, we administered it orally either to intact rats or to castrated rats, supplemented with T enanthate. Non-hypercalcemic doses of BXL-353 reduced time- and dose-dependently the androgen effect on ventral prostate weight, similarly to finasteride. Comparable results were obtained after chronic administration of BXL-353 to intact rats. Clusterin (an atrophy marker) gene and protein were up-regulated by BXL-353 in rat prostate, and nuclear fragmentation was widely present. The anti-androgenic properties of BXL-353 did not interfere with pituitary and testis function, as assessed by serum determination of rat LH and T. BXL-353 did not compete for androgen binding to BPH homogenates and failed to inhibit 5 reductase type 1 and type 2 activity. In conclusion, BXL-353 blocks in vitro and in vivo androgen-stimulated prostate cell growth, likely acting downstream the AR, without affecting calcemia, or sex hormone secretion. BXL-353 and other vitamin D₃ analogs might thus represent an interesting class of compounds for treating patients with BPH.