Fibroblasts isolated and expanded from ReLEx SMILE lenticules can be a source of human keratocytes. Since corneal keratocytes are quiescent cells, it is difficult to expand them in vitro in suitable numbers for clinical and experimental use. In the present study, this problem was solved by isolating and growing corneal fibroblasts (CFs) with a high proliferative potential and their reversion to keratocytes in a selective serum-free medium. Fibroblasts reversed into keratocytes (rCFs) had a dendritic morphology and ultrastructural signs of activation of protein synthesis and metabolism. The cultivation of CFs in a medium with 10% FCS and their reversion into keratocytes was not accompanied by the induction of myofibroblasts. After reversion, the cells spontaneously formed spheroids and expressed keratocan and lumican markers, but not mesenchymal ones. The rCFs had low proliferative and migratory activity, and their conditioned medium contained a low level of VEGF. CF reversion was not accompanied by a change with the levels of IGF-1, TNF-alpha, SDF-1a, and sICAM-1. In the present study, it has been demonstrated that fibroblasts from ReLEx SMILE lenticules reverse into keratocytes in serum-free KGM, maintaining the morphology and functional properties of primary keratocytes. These keratocytes have a potential for tissue engineering and cell therapy of various corneal pathologies.