Introduction: "Hypofunctional teeth" refer to the teeth which are not in intercuspation, such as high-positioned canines, open bite incisors, and impacted teeth. Compared with normal teeth, hypofunctional teeth have more periodontal ligament (PDL) atrophy. Previous studies have suggested that application of orthodontic force before extraction increases PDL width, eases the extraction, and may prevent root resorption after replantation. However, the effect of orthodontic tooth movement on the enhancement of human PDL tissue of hypofunctional tooth has never been investigated. Objectives: To examine the amount of PDL tissue on the root surface of extracted third molars following pre-application of orthodontic force Materials and Methods: The subjects were thirteen orthodontic patients who required third molar extraction before or during the treatment and had mesio-angular impacted mandibular third molars on one side and mandibular third molars which can be removed by simple extraction on another side. The experimental mesio-angular impacted third molars (n=13) received orthodontic force using a special appliance, while the contralateral third molars (n=13) received no force application as a control. Alteration in angulation and position of the third molars were assessed by panoramic radiographs. The extracted teeth were stained with toluidine blue to determine the amount of remaining PDL on the root surface under stereomicroscopy. The percentages of stained PDL areas were then compared between the experimental and control groups. Results: The mean angulation of experimental third molars was decreased to 54.4 (± 16.3) % of that before applying force. Mean duration of the orthodontic force application was 14.6 (± 8.1) weeks. Moreover, the mean percentage of PDL tissue on preloaded teeth was significantly higher than that on unloaded teeth (P 0.05). Conclusions: The application of orthodontic force leads to tooth movement, which consequently increases the amounts of PDL tissue in impacted third molars. Thereby, this study may propose the effect of orthodontic tooth movement on the improvement of the PDL quality in the hypofunctional tooth. Keywords: hypofunctional teeth, third molars, orthodontic loading, periodontal ligament