We investigated the effects of low-intensity pulsed ultrasound (LIPUS; 30 mW/cm2 spatial and temporal average) on the timing of LIPUS treatment in distraction osteogenesis. Lengthening of the right tibia was performed in 75 male Japanese white rabbits using unilateral fixators (waiting period, 7 days; distraction rate, 1.5 mm/day; distraction period, 7 days). Rabbits were divided into four groups according to the timing of the LIPUS treatment. Control group had no stimulation. Waiting group was treated with a daily 20-min session of LIPUS during a 7-day latency period. Lengthening group was treated during the lengthening period. Maturation group was treated for the first 7 days after completion of distraction. We evaluated the distraction site by radiography and histology every week for 4 weeks. Bone mineral density (BMD) and mechanical strength were tested and microfocus X-ray computed tomography was performed on specimens 2 weeks after completion of distraction. The lengthening group had greater BMD and mechanical strength than the other groups, bone regeneration was enhanced more in the maturation group than in the control or waiting groups. Histologically, endochondral bone formation in the lengthening and the maturation groups occurred earlier than in the control or waiting groups. These results suggest the LIPUS effect is mediated via endochondral pathways. We concluded that LIPUS stimulates bone formation in distraction osteogenesis and is most effective during the lengthening phase.