AbstractPurpose To evaluate the effects of transepithelial corneal crosslinking (TE‐CXL) on epithelium and stroma in human corneasMethods Fifteen corneal buttons were examined. Ten were from patients with keratoconus submitted to penetrating keratoplasty (PKP). Five of them were treated with TE‐CXL 2 hours before PKP, five of them were treated with TE‐CXL 3 months before PKP. Five normal corneal buttons from healthy donors were used as controls. TE‐CXL was performed with two different time of imbibition: 30 minutes and 2 hours.All samples were prepared for the detection of keratocyte apoptosis by TUNEL assay and for the morphological evaluation of epithelium and stroma by immunohistochemical analysis (Connexin 43, CD34).Results Normal corneas exhibited no TUNEL positive keratocytes while keratoconus and crosslinked samples showed moderate apoptotic cells in the anterior part of the stroma. Moreover, the samples treated with TE‐CXL 2 hours before PKP showed also an almost completely deteriorated epithelium with TUNEL positive cells. The epithelial positivity for connexin43 (transmembrane protein that forms gap junction channels) was similar in the control and in the corneas with crosslinking 3 months before PKP, while seemed more scattered in the keratoconus. In the samples treated with TE‐CXL 2 hours before PKP the positivity was patchy in the few remained epithelial cells.Conclusion The treatment with TE‐CXL leads to epithelial damage and a reduction of keratocytes in the sub‐epithelial region in the corneas treated 2 hours before PKP. In the samples treated with TE‐CXL 3 months before PKP the positivity of both CD34 keratocytes and connexin‐43 epithelial cells is similar to control.