BACKGROUND: The fat mass and obesity-associated protein (FTO) is an RNA demethylase that contributes to several physiological processes. Nonetheless, the impact of FTO on bone remodeling in the midpalatal suture while undergoing rapid maxillary expansion (RME) remains unclear. METHODS: First, to explore the expression of FTO in the midpalatal suture during RME, six rats were randomly divided into two groups: Expansion group and Sham group (springs without being activated). Then, suture mesenchymal stem cells (SuSCs) were isolated as in vitro model. The expression of FTO was knocked down by small interfering RNA to study the effect of FTO on the osteogenic differentiation of SuSCs. Finally, to evaluate the function of FTO in the process of bone remodeling in the midpalatal suture, ten rats were randomly divided into two groups: FB23-2 group (10 μM, a small molecule inhibitor of FTO) and DMSO group (control). RESULTS: Increased arch width and higher expression of OCN and FTO in the midpalatal area were observed in expansion group (P < .05). In the in vitro model, the mRNA expression levels of Runx2, Bmp2, Col1a1, Spp1, and Tnfrsf11b were decreased (P < .05) upon knocking down FTO. Additionally, the protein levels of RUNX2 and OPN were also decreased (P < 0.05). Adding FB23-2, a small-molecule inhibitor targeting FTO, to the medium of SuSCs caused a decrease in the mRNA expression levels of Runx2, Bmp2, Col1a1, Spp1, and Tnfrsf11b (P < 0.05). There was a statistically significant difference in evaluating the expression of OCN and OPN on the palatal suture between the FB23-2 and DMSO groups (P < .05). LIMITATION: The molecular mechanisms by which FTO regulates SuSCs osteogenesis remain to be elucidated. The FTO conditional knock out mouse model can be established to further elucidate the role of FTO during RME. CONCLUSION: FTO contributes to the osteogenic differentiation of SuSCs and plays a promoting role in midpalatal suture bone remodeling during the RME.