Background: The biomechanical property of the periodontal ligament (PDL) is important in orthodontics and prosthodontics. The objective of this study was to evaluate the feasibility of measuring the biomechanical behavior of the periodontal ligament using micro-computed tomography (micro-CT). Methods: A custom-made apparatus measured the force and displacement of a porcine PDL specimen within the micro-CT environment. Synchronized computed tomography (CT) images were used to obtain the deformation and displacement of the entire specimen and to reconstruct the three-dimensional mesh model. To match the experimental results, finite element analysis was then applied to simulate the biomechanical response of the PDL. The mechanical model of the PDL was assumed as the hyperelastic material in this study. Results: The volume variations of the tooth and the alveolar bone were less than 1%, which implies that tooth displacement was caused mostly by displacement of the PDL. Only translational displacement was observed with each load step because the transformation matrix acquired from the CT image registration was identical. The force-displacement curve revealed the nonlinear behavior of the PDL. There was a high correlation between the experimental displacement results and the simulation displacement results. The numerical results (based on the assumption that the PDL is the hyperelastic material) showed good agreement with the experimental results. Conclusions: Nondestructive measurements by micro-CT obtained the biomechanical behavior of the PDL. Using the hyperelastic characteristic as the constitutive model can properly predict the force-displacement relation of the PDL after loading. This study provided a feasible approach for measuring the biomechanical behavior of the PDL for further dental application.