AIM: To evaluate the differences in corneal biological parameters between transepithelial and epithelium-off corneal cross-linking in keratoconus. METHODS: In our prospective clinical trial, 40 patients (60 eyes) with progressive keratoconus were randomized to undergo corneal cross-linking with transepithelial (TE group, n=30) or epithelium-off (EO group, n=30) keratoconus. Examinations comprised topography, corneal biomechanical analysis and specular microscopy at 6mo postoperatively. RESULTS: The keratometer values were not significantly different between the TE and EO corneal cross-linked groups in different periods (each P>0.05). The corneal thickness of the EO group was greater than that of the TE group at 1wk after the operation (each P<0.05). Regarding corneal biomechanical responses, the EO group showed a longer second applanation length than TE group (P=0.003). Regarding the corneal endothelial function, standard deviation of the endothelial cell size, and coefficient of variation in the cell area, the values of EO group were larger than those of TE group at 1wk (P=0.011, 0.026), and the percentage of hexagonal cells in EO group was lower than that in TE group at 1 and 6mo (P=0.018, 0.019). CONCLUSION: Epithelium-off corneal cross-linking may strengthen corneal biomechanics better than TE procedure can. However, the TE procedure with a lower ultraviolet-A irradiation intensity would be safer for corneal endothelial function.