Purpose <p>To clarify the histological architecture of the intersphincteric region of the anal canal by delineating the layer-specific organization and spatial relationships among the anal sphincter complex and associated muscular and connective tissue components.</p> Methods <p>Tissue blocks containing the lateral wall of the anal canal were obtained from 11 adult human cadavers donated for anatomical research. Specimens were examined using descriptive histological and immunohistochemical analyses in transverse and coronal planes. The internal and external anal sphincters, longitudinal muscle, levator ani, interbundle gaps, and connective tissue compartments were identified and analyzed with respect to their three-dimensional organization.</p> Results <p>The intersphincteric region exhibited a heterogeneous and layered architecture rather than a uniform plane. The longitudinal muscle demonstrated a mosaic organization consisting of dense and loose components. The dense component terminated near the mid-height of the internal anal sphincter (mean, 54% of its length), whereas the loose component expanded inferiorly and formed a spacious compartment characterized by sparse smooth muscle fibers and loose connective tissue. Inferiorly, loose longitudinal muscle fibers branched and traversed natural interbundle gaps within the external anal sphincter. In addition, two partially overlapping layers of the levator ani were consistently observed, with interposed gaps contributing to the structural complexity of the intersphincteric region.</p> Conclusion <p>The intersphincteric region of the anal canal is a structurally complex and compartmentalized anatomical entity. Its heterogeneous histological architecture provides an anatomical substrate that may explain the initiation and directional spread of anal fistulas, including pathways described in classical fistula classifications.</p>