To investigate the effect, right timing, and mechanism of "accordion" technique on bone regeneration in rat distraction osteogenesis model.Fifty-four 12-week-old male Sprague Dawley rats underwent right tibial distraction osteogenesis procedure. After a 5-day latency, the distraction was performed for 7 days followed by 6-week consolidation. All animals were randomly divided into 4 groups based on different periods of "accordion" maneuvers in consolidation phase: control group ( n=18) with no manipulation, and three experimental groups including early-phase group ( n=18), mid-phase group ( n=12), and late-phase group ( n=6) with "accordion" maneuvers applied at 1, 3, and 5 weeks, respectively. The duration of the "accordion" maneuver was 7 days consisting of a 3.5-day compression and 3.5-day distraction. Rats in control group and early-phase group were sacrificed at 2, 4, and 6 weeks of the consolidation phase; rats in mid-phase group were sacrificed at 4 and 6 weeks of the consolidation phase; and rats in late-phase group were sacrificed at 6 weeks of the consolidation phase. Bilateral tibias from 6 rats in each group at each time point were obtained. Callus formation was monitored by X-ray radiography every week; new bone was reconstructed by Micro-CT three-dimensional reconstruction. The change of bone structure was evaluated, and parameters containing bone volume (BV)/tissue volume (TV) ratio (BV/TV) and bone mineral density (BMD) in three thresholds (158-211, 211-1 000, 158-1 000) were recorded and calculated at 6 weeks. Mechanical test consisting of ultimate load, modulus of elasticity, and energy to failure was performed. Histological analysis, such as Von Kossa staining, Safranin O staining, and HE staining, was done. Immunohistochemical staining using markers of osterix (OSX), osteocalcin (OCN), and vascular endothelial growth factor (VEGF) was analyzed.Images of X-ray showed that callus formation increased significantly in the mid-phase group. Micro-CT three-dimensional reconstruction demonstrated the mid-phase group owned fastest reconstructed speed among 4 groups, the cortical bone was continual at 6 weeks. At 6 weeks, the BMD and BV/TV in thresholds 158-1 000 and 211-1 000 in mid-phase group were higher than those in other groups. The results of mechanical test showed that ultimate load, modulus of elasticity, and energy to failure in mid-phase group were significantly higher than those in other groups ( P<0.05). Histological testing showed that the continuity of bone marrow cavity in mid-phase group was evident at 6 weeks after distraction. Immunohistochemical analyses confirmed the expression levels of osteogenesis (OCN, OSX) and angiogenesis (VEGF) elevated remarkably and then returned to normal in mid-phase group.The "accordion" technique is beneficial for new callus formation in distraction area. Applying the maneuver during the middle phase of the consolidation period was effective to accelerate new bone formation in rat distraction osteogenesis model.