Objectives: To investigate the effects of long-term, artificially created, hypofunctional occlusion and its recovery on the morphology of rat molar roots. Material and Methods: Eighteen 5-week-old Wistar-strain male rats were randomly divided according to their periodontal conditions into normal, hypofunctional, and recovery groups (n 5 6 in each). In the experimental hypofunctional and recovery groups, a bite-raising appliance was set to produce hypofunction at the molar region. All groups were analyzed at 16 weeks of age using three-dimensional micro-computed tomography. Root length, width, and area as well as the thickness and the area of the periodontal ligament (PDL) space of the maxillary first molar were calculated. Results: Roots were longer and narrower in the hypofunctional group than in the control group. The mesial root in particular showed a dramatic change. Root area also decreased significantly in the hypofunctional group compared to the other groups. Moreover, the PDL thickness and area decreased significantly in the hypofunctional group compared to the control group, but increased in the recovery group compared to the hypofunctional group. Conclusions: These findings suggest that root size and PDL structure may be reduced due to disuse atrophy resulting from a defect in occlusal function, but may be recovered following a gain of occlusal stimuli. (Angle Orthod. 2013;83:597-604.)