We investigated the therapeutic effects of a craniosynostosis-associated molecule, NEL-like molecule-1 (NELL1; NEL [a protein strongly expressed in neural tissue encoding the epidermal growth factor-like domain]), on osteolysis induced by polyethylene (PE)-particle debris. We used a murine calvarial osteolysis model with in vivo adenovirus (Ad)-mediated gene transfer. In total, 76 female Balb/c mice were randomly assigned to four groups for treatment 1 day postoperation: SHAM (injected with 0.1 mL saline without implantation of particles); PE control (injected with 0.1 mL saline after implantation of particles); PE+(Ad-GFP-NELL1) (injected with 0.1 mL Ad-GFP-NELL1 in saline after implantation of particles); and PE+(Ad-GFP) group (injected with 0.1 mL Ad-GFP in saline after implantation of particles). Green fluorescent protein (GFP) and NELL1 delivery in vivo after the injection were validated by optical imaging at 10 day postop, and then, all mice were sacrificed for analysis by three-dimensional (3D) microcomputed tomography (micro-CT), real-time polymerase chain reaction (PCR), histology, and biomechanical testing. Exogenous NELL1 and GFP were expressed in the osteolysis area for at least 9 days after the Ad-GFP-NELL1 injection. Serial 3D micro-CT images and testing of bone volume, bone mineral density, trabecular thickness, bone surface density, and connectivity density revealed that the new bone promoted with the Ad-GFP-NELL1 injection could almost compensate the PE-induced osteolysis and regenerate significantly better than with the Ad-GFP treatment. The expression of osteopontin (OPN) was significantly higher with Ad-GFP-NELL1 transduction among all the samples. Real-time PCR examination confirmed the augmented expression of OPN, Runx-2, and receptor activator of nuclear factor-kappa B ligand (RANKL). The elastic modulus was significantly greater with Ad-GFP-NELL1 than with the PE and/or Ad-GFP group (p<0.01). We found no transgene-associated toxic effects. Ad-GFP-NELL1 gene transfer effectively reversed the calvarial osteolysis and could be considered a new treatment for osteolysis through promoting bone regeneration.