To evaluate the effects of ascorbic acid (vitamin C), the main antioxidant agent in the cornea on transepithelial corneal cross-linking (CXL) where the main mechanism is oxidation.Twenty eyes of 20 rabbits were divided into 3 groups: Group 1 (7 eyes) had transepithelial corneal CXL after being fed with normal diet; Group 2 (7 eyes) had corneal CXL after once-daily subcutaneous injections of 200 mg of ascorbic acid in addition to normal diet; and the control group (6 eyes) was fed with normal diet but did not have corneal CXL performed. Ascorbic acid levels were measured in aqueous humor and plasma, and biomechanical measurements were applied to the cornea.There was a significant difference in ascorbic acid levels of plasma (P = 0.008) and aqueous humor (P = 0.006) between group 1 and 2. The Young's modulus values of group 1 and 2 were similar (P = 0.741) and were significantly higher than the control group (P = 0.02 and P = 0.01). The increase rate in Young's modulus values was 37.3% in group 1 and 43.9% in group 2 compared to control group. The ultimate strain values in group 1 and 2 were similar (P = 0.632) and were significantly higher than control group (P = 0.04, P = 0.03). The ultimate stress values in group 1 and 2 were similar (P = 0.836) and were significantly lower than control group (P = 0.001, P = 0.001).Systemic vitamin C does not appear to decrease effectiveness of transepithelial corneal CXL. Therefore, there is no reason to stop or reduce vitamin C supplementation before corneal CXL therapy.