The purpose of our study was to determine the impact of riboflavin-UVA photodynamic inactivation (PDI) (Collagen crosslinking technique) on viability, cell cycle phase, apoptosis and proliferation of human corneal endothelial cells (HCECs), in vitro. A HCEC line was cultured in DMEM/Ham's F12 medium supplemented with 5% fetal calf serum. HCECs cultures underwent 370 nm-UVA-light illumination for 4.1 minutes during exposure to 0.05% or 0.1% riboflavin and 20% dextran containing PBS. Twenty-four hours after riboflavin-UVA-PDI, viability was determined by the Alamar blue assay, cell cycle phase and apoptosis of the cells using the APO-DIRECTTM Kit, and two and twenty-four hours after PDI, HCECs proliferation by the BrdU Cell Proliferation Assay Kit. Twenty-four hours after the use of 0.1% riboflavin concentration without illumination and after 0.05% and 0.1% riboflavin-UVA-PDI, HCECs viability decreased significantly (P<0.01 for all) compared to controls. Twenty-four hours following riboflavin-UVA-PDI, the percentage of HCECs at the G1 cell cycle phase decreased significantly using 0.05% or 0.1% riboflavin concentration (P=0.02 and P=0.03), the percentage of HCECs at the G2/M phase increased significantly using 0.05% riboflavin concentration (P=0.03), compared to controls. Two and twenty-four hours after riboflavin-UVA-PDI using 0.05% or 0.1% riboflavin concentration, HCEC proliferation decreased significantly (P=0.02 for all). There was no significant difference in percentage of apoptotic HCECs at any of the treated groups compared to controls 24 hours after riboflavin-UVA-PDI (P=0.10).Crosslinking arrests HCECs at the G2/M phase, decreases viability and proliferation, however does not trigger apoptosis of human corneal endothelial cells in vitro.