New bone formation is known to occur between the opened palatal bones after rapid mid-palatal expansion (RME), although the time-dependent changes in the mid-palatal suture after RME have not been fully examined. Thus, we investigated time-dependent morphological changes in the mid-palatal suture using in vivo micro-computed tomography (mCT) and the expression of bone morphogenetic factors. RME was performed by inserting a 1.5-mm-thick circular metal ring between the maxillary incisors of rats, and morphological changes in the mid-palatal suture were investigated using in vivo mCT imaging after RME. Bone morphogenetic protein 2 (BMP-2) and insulin-like growth factor-I (IGF-I) expression in the suture were also examined using reverse-transcription polymerase chain reaction and immunohistochemistry. The bone volume of the mid-palatal suture decreased after RME to a minimum of -0.34mm(3) on day 12, then increased with bone formation over time and reached -0.13mm(3) on day 24. Significant increases in BMP-2 and IGF-I mRNA expression after RME were found on day 3 compared with day 0. By immunohistochemistry, BMP-2 and IGF-I were detected in osteoblasts on days 5 and 7, in endothelial cells of blood vessels, and fibroblasts on day 7. Expansion of the mid-palatal suture continues for 12 days after a single RME, and restoration requires more than 30 days. Additionally, BMP-2 and IGF-I may play important roles in the restoration process.