Background The sphenoidal spine protrudes from the roof of the infratemporal fossa (ITF). This study aims to assess the anatomic relationships among the sphenoidal spine and other structures within the ITF from the perspective of an endoscopic endonasal access (EEA), and to explore the implications of these relationships. Methods An EEA to the ITF was completed on six cadaveric specimens (12 sides). The anatomical relationships among the sphenoidal spine and adjacent structures were explored and associated distances from each other were measured using a navigation system. Results The foramen spinosum is located anterosuperior to the sphenoidal spine, whereas the chorda tympani courses caudal and medial to the sphenoidal spine and the Eustachian tube and parapharyngeal internal carotid artery (pICA) are at its posterior aspect. Two virtual vertical planes, at the anterior and posterior aspects of the sphenoidal spine, respectively, correspond to the posterior trunk of V-3 and middle meningeal artery, and the stylopharyngeal aponeurosis. The average length of sphenoidal spine was 8.5 +/- 2.43 mm, and the distance from distal apex of the sphenoidal spine to the foramen ovale, foramen spinosum, and pICA were 10.82 +/- 0.83 mm, 6.42 +/- 0.52 mm, and 5.02 +/- 0.54 mm, respectively. Conclusions The sphenoidal spine is a meaningful landmark for endonasal approaches to the ITF. Measurements and conceptualization of vertical planes prior and posterior to the sphenoidal spine are beneficial to better appreciate the anatomic relationships in the ITF.