The nasolabial fold is known to be a challenging midface feature for aesthetic physicians. However, the steric conformation of the structures related to the nasolabial fold has remained undefined because the composition and topography of this region are highly intricate. Therefore, this study aimed to clarify the three-dimensional structures of the nasolabial fold using micro-computed tomography and verify their detailed composition by means of histologic observation. Twenty-four specimens were collected from the area beside the alae nasi to the area above the oral angle of 12 cadavers (mean age, 80.3 years) bilaterally. Twelve specimens were evaluated by means of phosphotungstic acid contrast staining, and the rest were evaluated by means of histologic staining. All specimens were divided into three regions and analyzed comprehensively. The medial region of the nasolabial fold had dense irregular connective tissue intermingled with muscle fibers; the lateral region of the nasolabial fold had numerous fibrous septa with abundant adipose tissue. The levator labii alaeque nasi and the zygomaticus minor were attached to the medial part of the nasolabial fold, and the fascial septa were intermittently tethered to the dermis, lateral to the nasolabial fold. The extension of the adipose tissue within the fascial septa was limited by the lateral border of the muscle attachment. Dimensional and distributional alterations of the adipose tissues with senescence could render the nasolabial fold deeper by increasing the depth of the subcutaneous layer, lateral to the fold. Thus, to ameliorate the fold, the adipose tissue, lateral to the fold, or the muscle traction, medial to the fold, should be altered.