Negative pressure wound therapy (NPWT) has been recognized as an effective method for the treatment of intractable wounds. However, its effects on bone healing remain to be elucidated. Our previous study demonstrated that NPWT induced cell proliferation and osteoblastic differentiation of rat periosteum-derived mesenchymal stem cells (P-MSCs). It was reported that following NPWT treatment, the expression of the mechanotransduction molecule integrin beta 5 is increased, indicating that NPWT may serve an active role in fracture healing by enhancing bone formation and reducing bone resorption. The present study sought to further investigate the efficacy of NPWT on the bone regeneration process in a rabbit radial gap-healing model. All rabbits with radial defects were randomly divided into two groups: NPWT and control groups. Continuous negative pressure at -125 mmHg was applied to all rabbits. Furthermore, X-ray imaging and scoring on day 7, 14, 21 and 28 postoperatively were performed to evaluate new bone formation. Histological changes were determined via hematoxylin and eosin and Masson's trichrome staining at 2- and 4-weeks following surgery. In addition, vimentin-positive cells located in the periosteum were detected via immunohistochemical examination on day 3 post operation. Finally, protein expression levels of vascular endothelial growth factor (VEGF), bone morphogenetic protein (BMP)-2 and osteopontin (OPN) were evaluated using western blot analysis on the 2nd and 4th week following NPWT. X-ray and histological examination revealed that the bone-healing processes in the NPWT group were faster compared with the control group. Additionally, compared with the control group, the NPWT group exhibited higher X-ray scores, increased percentage of positive vimentin-stained cells and upregulated expression of VEGF, BMP-2 and OPN proteins. The aforementioned findings suggest that NPWT, under a continuous negative pressure of -125 mmHg, may accelerate bone regeneration by enhancing MSC proliferation, osteoblastic differentiation and VEGF, BMP-2 and OPN expression.