Background: The purpose of using this three-dimensional con-struct is to make a sufficient thickness membrane as a part of the uterine endometrium to protect implantation of the zygote. Materials and Methods: In this study, Polycaprolactone/ Gelatin/Polydimethylsiloxne (PCL/G/PDMS) nanofibers with aligned and random fiber arrangements were used as models to study the growth of uterine endometrial cells. To imitate in vivo tissue structure, three-dimensional (3D) cell/micro-nanofiber constructs with cells embedded among micro-nanofiber layers were built via layer-by-layer assembly. This structure showed that aligned nanofibers in the 3D constructs continuously in-duced cell polarization and promoted and demonstrated the po-tential of 3D cell/nanofiber construct as a model for prolifera-tion of uterine endometrial cells in a physiologically relevant environment. A layer-by-layer assembly approach was adopted to stack cell-seeded nanofiber mesh (aligned or random) into 3D multilayered constructs. Briefly, aligned and random na-nofibers collected on an aluminum foil were evenly seeded with endometrial cells (106 cells/scaffold). After 24 hrs, ten layers of cell-seeded nanofiber meshes were overlaid to each other. Fol-lowing this approach, one type of 3D cell/nanofiber constructs with either aligned or random nanofibers were formed. To visu-alize cell morphology inside the 3D cell/nanofiber constructs, at days 7 and 14 after seeding, samples were fixed in 4% glutaral-dehyde and cut into 5-μm thin sections. The sections were then stained with hematoxylin and eosin (H&E) to evaluate the cell distribution. SEM micrographs of the scaffolds were obtained before seeding of the cells.Results: Histological results showed that the cells homogene-ously distributed through the entire constructs, and formed an integrated connection with nanofibers. Cells cultured on 2D random nanofiber meshes exhibited a polygonal morphology. Similar morphology arrangements were also observed with the cells cultured in 3D sandwich constructs.Conclusion: A layer-by-layer approach was taken to assemble cell-seeded nanofiber meshes into 3D constructs with precisely controlled organization of nanofibers for mimicking the isotro-py (i.e., stacking random nanofiber layers) of uterus tissue. This 3D culture system allows us to understand nanofiber-induced cellular responses in a physiologically relevant environment and preparation for a proper engineered tissue.