© 2020, The Author(s). Periodic myometrial contraction is one of the important uterine functions to achieve embryo implantation and parturition. Although it is well-known that the mammalian myometrium is composed of longitudinal (outer) and circular (inner) layers, the precise mechanisms that coordinate both muscular contractions to produce peristaltic movements remain unclear. Recently, by treatment with our modified Clear Unobstructed Brain Imaging Cocktails and Computational analysis (CUBIC) tissue-clearing method, we obtained well-contrasted three-dimensional images of the transparent murine ovary using enhanced green fluorescent protein (EGFP) transgenic mice and light-sheet microscopy. Consequently, to investigate accurate anatomical connections between outer and inner myometrial fibers, we observed whole structures of the myometrium using a transparent murine uterus. By this method, we identified a novel muscle layer, a middle layer of the myometrium, which anatomically connects the conventional outer longitudinal and inner circular muscles. This new layer was visualized as a mesh-like structure and this structure was observed throughout the whole uterus from proximal to distal sites. In this area, CD31-positive vessels were abundantly localized around the mesh-like muscle fibers. In addition, CD34-positive uterine telocytes and tubulin β-3-positive nerve fibers were closely located in this middle layer. These findings indicate the presence of a novel mesh-like stratum that connects longitudinal and circular muscle layers, and suggest its coordinating role in myometrial contractions.