In the present study, we found that two hormone receptor-positive human breast cancer cell lines, ZR-75-1 and BT-474, naturally expressed steroid sulfatase (STS) protein and had catalytic activity to produce estrone from estrone sulfate (E1S) with a comparable level to those in human breast cancer tissues. E1S at physiological concentrations stimulated the growth of those cells. A novel steroidal STS inhibitor, KW-2581 inhibited the STS activity of ZR-75-1 cells with an IC(50) of 13 nM, a potency equal to or higher than that of the non-steroidal STS inhibitor, 667 COUMATE. The inhibitory effect of KW-2581 was enhanced by pre-incubation with STS enzyme, suggests being irreversible inhibition. KW-2581 inhibited the E1S-stimulated growth of ZR-75-1 cells with an IC(50) of 0.18 nM, but failed to inhibit the growth stimulated by 17beta-estradiol. Expression of E1S-induced progesterone receptors in ZR-75-1 cells was reduced by treatment of KW-2581 at concentrations as low as 0.1 nM. Oral administration of KW-2581 for 4 weeks caused tumor shrinkage in a mouse xenograft model. Tumor STS activity had been completely (>95%) eliminated by 24 hours after the last administration. These findings suggest that KW-2581 has considerable potential for therapeutic development as a novel anti-hormonal drug for treatment of breast cancer.