Periodontitis is a common oral disease characterized by progressive destruction of periodontal tissue and loss of teeth. However, regeneration of periodontal tissue is a time-consuming process. Low-intensity pulsed ultrasound (LIPUS) is a widely used non-invasive intervention for enhancing bone healing in fractures and non-unions. With the hypothesis that LIPUS may accelerate periodontal regeneration, the effects of LIPUS on periodontal tissue regeneration were investigated both in vitro and in vivo. LIPUS (90 mw/cm2, 20 min/d, 1.5 MHz) was applied to stimulate dog periodontal ligament cells (dPDLCS). The mRNA expression of BSP (P < 0.05), OPN (P < 0.05) and COL3 (P < 0.05) was increased significantly in the LIPUS group. The positive stained mineralized nodules by alizarin red in the LIPUS group were greater than in the control group (P < 0.05). Eight male beagle dogs were divided into 4 groups: guided tissue regeneration (GTR) group (G1), LIPUS + GTR group (G2), LIPUS group (G3), and control group (G4, no treatment). A 4 × 5 mm2 defect was created in the buccal alveolar bone. The modeling areas in the G2 and G3 groups were then exposed to LIPUS. Eight weeks after surgery, histological assessment indicated increased periodontal tissue in the LIPUS + GTR group. Micro computed tomography (micro-CT) showed that the regenerated bone volume (BV) in the G2 was significantly higher than that in the G1, G3 and G4 groups (P < 0.05). The bone surface (BS) trabecular number (Tb.N) and trabecular thickness (Tb.Th) in G2 were markedly higher than in G4 (P < 0.05). It is concluded that LIPUS + GTR can accelerate new alveolar bone formation, with a prospective for promoting periodontal tissue repair.