Selective inhibitors of cathepsin K, which has a major role in the degradation of bone collagen, are potential new treatments for osteoporosis. The pharmacokinetics and the pharmacodynamic effects on biochemical markers of bone turnover of the new cathepsin K inhibitor, ONO-5334, were investigated in a multiple ascending dose, phase 1 study. A total of 120 healthy postmenopausal women were enrolled, and doses of 10 to 600 mg once daily and 50 and 300 mg twice daily were evaluated in 15- and 28-day multiple-dosing cohorts. Plasma ONO-5334 concentration reached steady state within 2 days. Twenty-four hours after the last dose in the 15-day multiple-dose cohort, 100, 300, and 600 mg once daily reduced urinary C-terminal telopeptide of type I collagen by a mean (± standard deviation) 44.9% ± 13.6%, 84.5% ± 4.4%, and 92.5% ± 1.3%, respectively. The 28-day cohort showed similar effects. There were far smaller effects on bone-specific alkaline phosphatase (B-ALP), tartrate-resistant acid phosphatase 5b (TRAP5b), or osteocalcin (OC) (measured after 28 days). ONO-5334 was well tolerated up to 600 mg/d and for up to 28 days of multiple dosing. Multiple dosing with ONO-5334 100 mg resulted in considerable suppression of bone resorption markers with no appreciable effects on bone formation markers (B-ALP, OC) or osteoclast number (TRAP5b).