Spherical culture could promote the plasticity and stemness of human corneal stromal cells (hCSCs). Here, we introduce a novel three-dimensional (3D) cell culture system based on a polydimethylsiloxane (PDMS) microwell platform composed of many V-bottom microcavities to generate human corneal stromal cell spheroids and promote cell stemness. We isolated hCSCs from SMILE-derived lenticules and maintained their physiological phenotype by culturing them in a medium supplemented with human corneal stromal extract (hCSE). Utilizing a PDMS microwell platform fabricated through 3D printing technology, we successfully generated 3D corneal stromal cell spheroids (3D-CSC) with uniform size and stable structure, exhibiting increased expression of pluripotency factors, including OCT4, NANOG, SOX2, KLF4, and PAX6. Furthermore, the iPS supernatant of E8-conditioned medium (E8-CM) significantly enhanced the stemness properties of these cells. RNA sequencing and proteomics analyses revealed that 3D-CSCs exhibited superior proliferation, differentiation, cell adhesion, migration, and neurogenesis compared to traditional monolayer cultures, underscoring the role of biophysical cues in promoting hCSCs stemness. In summary, this study presents an effective 3D cell culture platform that mimics the in vivo microenvironment, facilitating the enhancement of stemness properties and providing valuable insights into corneal tissue engineering and regenerative medicine, particularly for treating corneal opacities and diseases.