Abstract Objectives The mid-palatal expansion technique is commonly used to correct maxillary constriction in dental clinics. However, there is a tendency for it to relapse, and the key molecules responsible for modulating bone formation remain elusive. Thus, this study aimed to investigate whether signal transducer and activator of transcription 3 (STAT3) activation contributes to osteoblast-mediated bone formation during palatal expansion and relapse. Methodology In total, 30 male Wistar rats were randomly allocated into Ctrl (control), E (expansion only), and E+Stattic (expansion plus STAT3-inhibitor, Stattic) groups. Micro-computed tomography, micromorphology staining, and immunohistochemistry of the mid-palatal suture were performed on days 7 and 14. In vitro cyclic tensile stress (10% magnitude, 0.5 Hz frequency, and 24 h duration) was applied to rat primary osteoblasts and Stattic was administered for STAT3 inhibition. The role of STAT3 in mechanical loading-induced osteoblasts was confirmed by alkaline phosphatase (ALP), alizarin red staining, and western blots. Results The E group showed greater arch width than the E+Stattic group after expansion. The differences between the two groups remained significant after relapse. We found active bone formation in the E group with increased expression of ALP, COL-I, and Runx2, although the expression of osteogenesis-related factors was downregulated in the E+stattic group. After STAT3 inhibition, expansive force-induced bone resorption was attenuated, as TRAP staining demonstrated. Furthermore, the administration of Stattic in vitro partially suppressed tensile stress-enhanced osteogenic markers in osteoblasts. Conclusions STAT3 inactivation reduced osteoblast-mediated bone formation during palatal expansion and post-expansion relapse, thus it may be a potential therapeutic target to treat force-induced bone formation. midpalatal mid clinics However relapse elusive Thus STAT (STAT3 osteoblastmediated osteoblast mediated total control, control , (control) only, only only) EStattic STAT3inhibitor, STAT3inhibitor STATinhibitor inhibitor, inhibitor STAT3-inhibitor Microcomputed Micro computed tomography 14 10% 10 (10 magnitude 05 0 5 0. frequency 2 duration inhibition loadinginduced loading induced ALP (ALP) blots COLI, COLI COL I, I COL-I Runx2 Runx osteogenesisrelated osteogenesis related Estattic stattic forceinduced force attenuated demonstrated Furthermore stressenhanced enhanced postexpansion post (STAT (control 1 (1 (ALP (