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OBJECTIVES Positive effects of irisin on osteogenic differentiation of periodontal ligament (PDL) cells have been identified previously, this study aims to examine its effect on orthodontic tooth movement (OTM) in vivo. MATERIALS AND METHODS The maxillary right first molars of male Wistar rats (n = 21) were moved mesially for 14 days, with submucosal injection of two dosages of irisin (0.1 or 1 μg) or phosphate-buffered saline (control) every third day. OTM was recorded by feeler gauge and micro-computed tomography (μCT). Alveolar bone and root volume were analysed using μCT, and plasma irisin levels by ELISA. Histological characteristics of PDL tissues were examined, and the expression of collagen type I, periostin, osteocalcin (OCN), von Willebrand factor (vWF) and fibronectin type III domain-containing protein 5 (FNDC5) in PDL was evaluated by immunofluorescence staining. RESULTS Repeated 1 μg irisin injections suppressed OTM on days 6, 9, and 12. No significant differences were observed in OTM in the 0.1 μg irisin group, or in bone morphometric parameters, root volume or plasma irisin, compared to control. Resorption lacunae and hyalinization were found at the PDL-bone interface on the compression side in the control, whereas they were scarce after irisin administration. The expression of collagen type I, periostin, OCN, vWF, and FNDC5 in PDL was enhanced by irisin administration. LIMITATIONS The feeler gauge method may overestimate OTM. CONCLUSIONS Submucosal irisin injection reduced OTM by enhancing osteogenic potential of PDL, and this effect was more significant on the compression side.