Osteoprotegerin (OPG) is a naturally occurring protein, which prevents bone resorption by inhibition of osteoclastogenesis, function, and survival. Therefore, recombinant OPG may be an attractive drug in the treatment of chronic bone resorptive diseases such as osteoporosis. Gene therapy has the potential to achieve long-term treatment by delivering genes of anti-resorptive proteins to the recipient. The effects of OPG gene therapy on fracture healing have not been described previously. The influence of OPG gene therapy on callus formation, callus tissue structural strength, apparent material properties, and histology of tibia fractures in rats was investigated after 3 weeks and 8 weeks of healing. Intramuscular administration of adeno-associated virus (AAV) vector-mediated OPG resulted in increased levels of OPG in serum of approximately 100 ng/ml throughout the study period. Control animals with fractures received transduction with an AAV reporter gene construct (AAV-enhanced green fluorescent protein (eGFP)), and in this group serum OPG levels remained at baseline (<10 ng/ml). After 3 weeks of healing, AAV-OPG treatment reduced the number of osteoclasts in the callus tissue (33%, P < 0.001). However, AAV-OPG treatment did not influence callus dimensions, callus bone mineral content (BMC), fracture structural strength, or apparent callus tissue material properties. After 8 weeks of healing, AAV-OPG treatment reduced the number of osteoclasts in the callus tissue (31%, P < 0.001) compared with AAV-eGFP fractures. Furthermore, deposition of new woven bone at the fracture line of the original cortical bone was hampered (new woven bone present: in all AAV-eGFP animals, in 41% of AAV-OPG-treated animals, P < 0.001). AAV-OPG treatment also increased callus BMC (18%, P = 0.023) compared with AAV-eGFP fractures. AAV-OPG did not influence callus dimensions, structural strength of the fractures, or ultimate stress, whereas elastic modulus was reduced in the AAV-OPG groups (37%, P = 0.039). The experiment demonstrates that AAV-OPG gene therapy decreases the fracture remodeling, but this does not influence the structural strength of healing fractures.