In vivo three-dimensional (3D) kinematics of the lumbar spine has not been well evaluated by the conventional methods because of their methodological limitations, while 3D intervertebral motions have been quantitatively determined by cadaver studies. We thus developed a novel 3D analyzing system for the relative motions of individual vertebrae using 3D magnetic resonance imaging (MRI) and analyzed in vivo 3D intervertebral motions of the lumbar spine during trunk rotation. Ten healthy volunteers underwent 3D MRI of the lumbar spine in nine positions with 15 degrees increments during trunk rotation (0 degrees , 15 degrees , 30 degrees , 45 degrees , and maximum). Relative motions of the lumbar spine were calculated by automatically superimposing a segmented 3D MRI of the vertebra in the neutral position over images of each position using the voxel-based registration method. These 3D motions were represented with 6 degrees of freedom by Euler angles and translations on the coordinate system. The mean axial rotation of ten healthy volunteers of each lumbar spinal segment in 45 degrees trunk rotation to each side ranged from 1.2 degrees to 1.7 degrees . Coupled flexion with axial rotation was observed at the segments from L1/2 to L5/S1. Coupled lateral bending of the segments from L1/2 to L4/5 was in the opposite direction of the trunk rotation, while that of T12/L1 and L5/S1 was in the same direction. The direction of the coupled lateral bending in the present study was different from that in the previous cadaver study only at L4/5. This difference might result from the non-load state of the supine position in the current study and/or the non-physiological state in the cadaver study. Our system has two limitations: (1) the study was conducted with each volunteer in the supine position, and (2) because the rotation device regulated trunk rotation, trunk rotation might not have been physiological. In vivo 3D intervertebral motions of the lumbar spine during trunk rotation were evaluated using our novel motion analysis system. These data may be useful for the optimal orthopaedic management of lumbar spinal disorders.