The periosteum is a rich source of osteoprogenitor cells and periosteal grafts can be used as an alternative method to replace bone grafts. The low-intensity pulsed ultrasound (LIPUS) has often been used as a noninvasive method to stimulate osteogenesis and reduce the fracture healing time. The aim of this study was to evaluate the effects of the ultrasound exposure on the rat tibia periosteum. Group I (7 animals) received LIPUS therapy on the left tibia for 7 days and group II (7 animals) on the left tibia for 14 days. After euthanasia, the tibias were processed. Number of periosteal cells and vessels and thickness of the periosteum were analyzed. The number of periosteal cells was higher in stimulated periosteum compared to controls at 7 and 14 days, but the number of vessels and the thickness only were higher in the group stimulated at 14 days. Furthermore, the ultrasound treatment for 14 days was more effective than 7 days. The ultrasound stimulation of the periosteum prior to grafting procedure can be advantageous, since it increases periosteal activity, and LIPUS may be an alternative method for stimulating the periosteum when the use of periosteal grafts in bone repair is needed.