Osteoporosis is a major health problem characterized by compromised bone strength that predisposes patients to an increased risk of fracture, more and more investigations are focusing on the treatment of osteoporotic fracture healing. However, there are few studies elucidating the efficacy of vitamin D, 1,25-dihydroxy vitamin D(3) (1,25(OH)(2)D(3)), on osteoporotic fracture healing. In the present study we have established an osteoporotic fracture rat model to evaluate the effects of 1,25(OH)(2)D(3) on fracture healing. Female SD rats of six-month-old (n=40) allocated randomly into two groups were given ovariectomy. Bilateral midshaft femoral osteotomy was performed 12 weeks post-ovariectomy. Then treatment was begun at the second day after osteotomy and continued until sacrifice at 6 and 16 weeks post-fracture with middle chain triglyceride (MCT) vehicle and 1,25(OH)(2)D(3) at 0.1 microg/kg/day by oral gavage. Fracture callus was evaluated by soft X-ray radiography, micro-computed tomography (micro-CT), biomechanical testing and histology. Soft X-ray radiography, at 6 weeks post-fracture, showed a less distinct fracture line in the 1,25(OH)(2)D(3) group compared with the MCT-vehicle group, however, the fracture line was invisible in both groups at 16 weeks post-fracture. Micro-CT based histomorphometric data, at 6 weeks post-fracture, showed that the total volume of callus (TV) was approximately 23% higher in the 1,25(OH)(2)D(3) group than that in the MCT-vehicle group (P<0.001), and the new bone volume (BV), BV/TV, the trabecular number (Tb.N), and density of TV also showed the same trend. At 16 weeks post-fracture, the increment still existed as shown by Tb.Th and density of TV (P<0.001, vs control). Biomechanical testing data, at 6 weeks post-fracture, showed that the ultimate load at failure and energy absorption of the 1,25(OH)(2)D(3) group were nearly one fold higher than that of the MCT-vehicle group (P<0.001). At 16 weeks post-fracture, the ultimate load and energy absorption were also higher with the treatment of 1,25(OH)(2)D(3) (P<0.01 vs control). Histology showed that the fracture callus in the 1,25(OH)(2)D(3) group was remodeled better compared to the control group. In conclusion, 1,25(OH)(2)D(3) could promote fracture healing by improving the histomorphometric parameters, mechanical strength and tendency to increase transformation of woven bone into lamellar bone in an ovariectomized rat model.