The location of the instantaneous helical axis (IHA) and the impact of the facet joints (FJ) on the kinematics in the thoracic spine remain inconclusive. This study aimed to examine the IHA in the functional spinal unit (FSU) T4-5 during axial rotation in intact conditions and after bilateral facetectomy. Four human T4-5 FSUs were examined with an established 6D measuring apparatus in intact conditions and after bilateral facetectomy. The IHA’s parameters migration, location, and direction in the horizontal plane were calculated. Defined preloads in different positions were applied. Under the intact conditions, the IHA migrated about 4 mm and from one to the contralateral side according to the applied preload. The location of the IHA was observed in the anterior part of the spinal canal. After bilateral facetectomy, the location of the IHA shifted ventrally about 10 mm compared to the intact conditions. Under intact conditions, the direction of the IHA was minimally dorsally reclined. After bilateral facetectomy, the IHA was significantly more ventrally inclined. The study determined the location of the IHA under intact conditions at the anterior part of the spinal canal. The IHA of the FSU T4-5 is substantially influenced by the guidance of the FJs.