The trajectory of the medial fibers of depressor anguli oris muscle that pass deep to the depressor labii inferioris muscle (dDAO) and the anatomic relationship have not yet been examined. The aim of this study was to clarify the arrangement of the dDAO and its relationships with the surrounding structures. The dDAOs were investigated in 40 hemifaces from Korean cadavers. The dDAOs were observed in 18 of the 40 specimens (45%). The anatomic relationships of the dDAO were classified into the following 4 categories according to their connection with the incisivus labii inferioris muscle (ILI): (1) connected to the ILI and attached to the inferior border of the mandible lateral to the mentalis muscle (14 specimens, 35%), (2) connected to the ILI and intermingled with the lateral lower fibers of the mentalis muscle (2 specimens, 5%), (3) connected to the ILI but neither attached to the mandible nor intermingled with the mentalis muscle (1 specimen, 2.5%), and (4) not connected to the ILI but intermingled with the mentalis muscle (1 specimen, 2.5%). Therefore, in 17 specimens, the dDAOs were connected to the ILI (42.5%), in 14 specimens, they were attached to the inferior border of the mandible (35%), and, in 3 specimens, they were intermingled with the mentalis muscle (7.5%). The new anatomic data regarding the dDAO will contribute toward the understanding of movements of the mouth and lower face, which will be helpful when designing effective botulinum toxin type A therapies and performing various types of facial surgery.