Introduction and Hypothesis <p>The urethra is surrounded by layers of smooth muscle, including inner longitudinal and outer circler muscles, as well as the skeletal muscle of the external urethral sphincter. However, the extent of these muscles and their relationship with the levator ani (LA) remain unclear. This study aimed to clarify the composition of muscle layers around the female urethra and their three-dimensional arrangements.</p> Methods <p>Pelvises from five female cadavers were collected for histological analysis. The surrounding urethral tissue was serially sectioned and stained with Elastica van Gieson stain to identify the connective and muscular tissues. Immunohistological staining with an anti-smooth muscle antibody was performed to confirm the distribution of the smooth and skeletal muscles. Thereafter, muscle layers were three-dimensionally reconstructed on the basis of serial histological sections.</p> Results <p>The female urethra was surrounded by four muscle layers arranged from inner to outer layers in the following order: longitudinal smooth muscle, circular smooth muscle, the external urethral sphincter (EUS), and an outermost smooth muscle layer. These muscles surrounded the urethra and partially extended anteriorly. Moreover, smooth muscle fibers extending from the vagina were found between the LA and EUS.</p> Conclusions <p>This study clarified the distribution of muscle tissue surrounding the female urethra, showing that the EUS is connected to the LA through the outermost smooth muscle layer. Thus, urination control likely involves both the urethral muscle layers and the pelvic floor muscles.</p>