Objective This study investigated the effect of local semaphorin 3A (Sema3A) administration on alveolar bone loss during OTM in a mouse model of periodontitis. Background Orthodontic tooth movement (OTM) for patients with periodontal disease is known to increase the risk of exacerbating alveolar bone loss due to inflammation of the periodontal tissue. However, its mechanism of action and prevention remains unclear. Methods Mice (male 7–8 weeks old, C57BL/6J, n = 12) were divided into six groups: untreated group (control), without OTM and recovered from induced periodontitis (RP), with OTM and administered PBS or Sema3A to the gingiva after induced periodontitis (VehPO, SemaPO), with OTM and administered PBS or Sema3A to the gingiva without periodontitis induction (VehNO, SemaNO). Samples were collected on 14 days, and bone loss, histological analysis, cytokine production level, and tooth movement were assessed. Cultured human periodontal ligament (hPDL) cells were stimulated with lipopolysaccharide (LPS) and compressive force (CF), and mRNA expression levels of Sema3A and its receptors were analyzed. Results The bone loss was significantly lower in the SemaPO group than in the VehPO group. The number of TRAP‐positive cells in the SemaPO group was significantly lower than that in the VehPO group and was at the same level as that in the control group. The receptor activator of nuclear factor (NF)‐kB‐ligand/osteoprotegerin (RANKL/OPG) ratio and the levels of proinflammatory cytokines, including interleukin (IL)‐1β, IL‐6, IL‐17, tumor necrosis factor (TNF)‐α, and interferon (IFN)‐γ, in the gingival tissues were significantly lower in the SemaPO group than in the VehPO group. Additionally, Sema3A mRNA expression in hPDL cells was significantly decreased by co‐stimulation with LPS and CF compared with that in the control group. Finally, the distance moved (dist.) and the mesial tipping angle (θ) was significantly smaller in the SemaPO group than in the VehPO group and was not significantly different from that of VehNO. Conclusion Pathological alveolar bone loss exacerbated by OTM in periodontitis might be prevented by local administration of Sema3A without inhibiting OTM.