Nitric oxide (NO) may modulate estrogen’s anabolic effects on bone homeostasis by restraining osteoclast-mediated bone resorption and stimulation of osteoblast activity. Accordingly, NO donated by organic nitrates, including nitroglycerin, is thought to protect against bone loss associated with estrogen deficiency. In this study, we have explored this phenomenon. Thirty-two 12-week-old female Wistar rats were divided into four groups prior to bilateral ovariectomy or a sham operation. The ovariectomised rats received (1) vehicle control (OVX control), (2) 17-β-estradiol (OVX+E2), or (3) transdermal nitroglycerin (OVX+NG) for 4 weeks. Femoral and tibial bone mineral density (BMD), serum alkaline phosphatase and urine deoxypyridinoline and NO metabolites were analysed at the end of the study period together with failure torque and torsional rigidity of the tibiae and cellular localisation of the NO-synthase (NOS) isoforms. In OVX+E2 group, proximal and distal femoral and proximal tibial BMD exceeded that of the Sham controls. Nitroglycerin prevented BMD loss at these three sites at levels comparable to that of the Sham controls. Deoxypyridinoline excretion did not change except in the OVX-E2 group that showed an expected reduction when compared to the Sham and OVX controls. There were no treatment-related differences in total alkaline phosphatase or urinary NO metabolites. Tibial failure torque was comparable between the groups but both OVX+E2 and OVX+NG groups showed decreased torsional rigidity compared with the OVX controls. Endothelial and inducible NOS were found in osteoblast-like cells associated with calcifying cartilage spicules in the distal femoral metaphysis. These data confirm previous findings and show that nitroglycerin counteracts the estrogen deficiency-induced osteopenia in the ovariectomised rat model. Organic nitrates may thus be beneficial in conditions where bone turnover is compromised such as in osteoporosis.