Background <p>Perioral rejuvenation is challenging due to the lack of spatial anatomical understanding of the labiomandibular fold (LMF). The LMF’s formation mechanism remains underexplored due to intricate relationships between musculature and subcutaneous structures. This study aimed to clarify the three-dimensional structures of the LMF using micro-computed tomography and histology.</p> Methods <p>Twenty-two rectangular specimens, following the LMF as the diagonal line, were retrieved bilaterally from eleven fresh frozen cadavers (mean age, 69.3 years). Eleven specimens were imaged with phosphotungstic acid contrast staining for micro-computed tomography, and the rest were histologically examined using Masson and Verhoeff-van Gieson staining. The LMF was divided into three equal-width regions for analysis.</p> Results <p>The LMF serves as a border between the lateral fat-abundant zone and the medial fat-scant zone statically. Dynamically, the LMF is accentuated with age by the downward pull of depressor muscles via a compact slanted collagen-elastin-muscle (CEM) network composed of the depressor anguli oris, platysma, and buccinator muscle fibers.</p> Conclusions <p>The LMF formation is governed by subcutaneous composition differences between the lower lip and cheek region and vertical dermal insertions of mimetic muscles. Perioral muscle contractions transmit directly at the skin via the CEM network, governing regional wrinkle severity and appearance. The micro-CT findings provide crucial three-dimensional insights into the layered architecture and intricate musculature correlation of the LMF region, providing regional-specific evidence for holistic rejuvenation practices.</p> Level of Evidence III <p>This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors <a href="http://www.springer.com/00266">www.springer.com/00266</a>.</p>