High mobility group protein B1 (HMGB1), a bone‐active cytokine and an osteocyte alarmin, might have dual functions in bone metabolism that could benefit bone formation and accelerate osteoclastogenic activity. High mobility group protein B1 was recently shown to be involved in tooth movement. Here, we investigated the expression of HMGB1, which remains poorly elucidated, under stress overload‐induced periodontal remodelling conditions in vivo. Thirty‐six Sprague‐Dawley rats (male, 180–200 g) were randomly divided into three groups: two experimental groups, in which 50 or 100 g of force was applied to the first molars for 7 d to induce movement; and one control group, in which no force was applied. These stresses induced tooth movement over significantly different distances, and marked morphological changes were consistently observed in the periodontal tissues of the experimental rats, as demonstrated by histological staining. A real‐time PCR analysis showed upregulation of the receptor activator of nuclear factor‐kappaB ligand‐to‐osteoprotegerin ratio and downregulation of the Hmgb1 gene. Changes in both location and expression of the HMGB1 were observed through immunofluorescence analysis. Our data suggest that HMGB1 expression during orthodontic tooth movement might be regulated in a time‐ and force‐dependent manner that is substantially more complex than anticipated.