Objectives To describe and document the anatomical and histological architecture of the vaginal outlet wall. Design A prospective observational case series study. Setting International centers. Population Twelve fresh human female cadavers. Methods Female corpses were subjected to stratum-by-stratum, macro-, and micro-anatomical dissections in sagittal, transverse, and coronal planes. Multiple excisional biopsies were obtained from the vaginal outlet wall at 12, 3, 6, and 9 o’clock locations for histological examination. Digital photograms documented the anatomical and histological features. Primary Outcome measures gross, topographic anatomy and characteristic histologic features of the vaginal outlet wall. Results The vaginal outlet wall (VOW) was positioned in the vertical orientation and fused with horizontally located the vaginal wall at the hymeneal plate level. The hymeneal membrane and ring were not attached directly to the vaginal wall. VOW has three identifiable anatomical-histological layers a) the hymeneal membrane, b) the hymeneal ring (cartilage) with different anatomy and histology than the vaginal wall, and c) the hymeneal plate (the deep level of VOW), which connects to the vaginal wall. Conclusions The vaginal outlet wall is an anatomical structure consisting of the hymeneal membrane, hymeneal ring (cartilage), and hymeneal plate. This structure is in vertical topographic orientation. Anatomically and histologically, it differs from the vaginal wall, which fuses with the deep layer of the hymeneal plate.