With recent advances in motion-sparing techniques in spine surgery, disc nucleus replacement (DNR) has been introduced as a viable method to restore the biomechanical functions of the spine. Several methods of DNR have been proposed in the literature. However, the risk of device migration or extrusion is a major issue that should be addressed for a successful DNR. DNR using a balloon nucleus (BN) filled with pressurized fluid may be capable of reducing such risks while preserving the advantages of DNR. The objective of this study was to investigate the biomechanical functionalities of the human cadaveric lumbar motion segments with a custom made BN filled with saline at internal fluid pressure of 0.3 or 0.6 MPa in terms of axial and rotational flexibilities of the L4-L5 motion segment. Axial flexibility was quantified by the axial displacement resulting from an axial compressive force of 400 N while the rotational flexibility by the range of motions determined as the rotational angles in response to a pure moment of 6.0 Nm in flexion, extension, and right- and left-lateral bending directions. These tests were performed successively on the motion segment in the following conditions: intact, post nucleotomy, implanting BN with 0.3 MPa, and BN with 0.6 MPa. The nucleotomy was found to significantly increase both the axial and rotational flexibilities while the implantation of the BN reduced the axial and rotational flexibilities to those of the intact segment. The axial and rotational flexibilities of the segment with the BN with 0.3 MPa were greater than those of the segment with the BN with 0.6 MPa. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:167-173, 2018.