The objective of the present study was to systematically explore the effects of 32K Da protein (32KP) on postmenopausal osteoporosis. Eighty 3-mo-old female Sprague-Dawley rats were employed and randomly divided into one sham-operated group (SHAM) and five ovariectomy (OVX) subgroups as OVX (control), OVX with 17-ethinylestradiol (E2, 25 g/kg/day), OVX with 32KP of graded doses (50, 50, or 150 mg/kg/day). 32KP or E2 diet was fed on week 4 after operation, for 16 weeks. Bone mass, bone turnover and strength were evaluated by dual-energy X-ray absorptiometry (DEXA), biochemical markers and three-point bending test, respectively. Femur marrow cavity was observed by light microscopy via hematoxylin-eosin staining. It is observed that different dosage treatment of 32KP increased the body weight and prevented the loss of bone mass induced by OVX. The prevention effect against bone loss was presumably due to the altering of the rate of bone remodeling. The bone mineral density and bone calcium content in OVX rats were lower than that in the control group, suggesting that 32KP was able to prevent significant bone loss. In addition, the data from three point bending test and femur sections showed that 32KP treatment enhanced bone strength and reduced the marrow cavity of the femur in OVX rats. In the serum and urine assay, 32KP decreased urinary deoxypyridinoline and calcium concentrations; however, serum alkaline phosphatase activities were not inhibited. It suggested that amelioration of bone loss was changed via inhibition of bone reabsorption. Our findings indicated that 32KP might be a potential alternative drug for the prevention and treatment of postmenopausal osteoporosis.