The origin of the posterior scrotal nerve is considered to be the bilateral pudendal nerves but the course to the midline is still obscure. Using 5 late-stage human male fetuses, we identified the single nerve through the intramuscular midline septum of the bulbospongiosus and the bilateral nerves along the left and right sides of the septum. Thus, the posterior scrotal nerve showed a variation: a single midline trunk or bilateral nerves. Branches of the bilateral pudendal nerves ran medially between the muscle and Cowper's gland and, at the midline area, they joined or associated closely. During the proximal course, much or less, the nerve penetrated the superior part of the muscle. The nerve entered the subcutaneous tissue at and near the perineal raphe. The communication with intrapelvic autonomic nerves were suggested behind Cowper's gland. Notably, the midline skin immediately anterior to the anus carried a considerable dense supply of thin sensory nerves. However, these nerves seemed to come from a space between the rectal smooth muscle and the external anal sphincter, not from the posterior scrotal nerve. Therefore, surgical treatment of the intersphincteric layer was likely to injure the original sensory nerve supply to the anterior anal skin.