Background: The effect of mitomycin-C (MMC) on the reduction of endothelial cell count in the cornea remains controversial. We aimed to evaluate the effect of MMC on corneal endothelial cell parameters after refractive surface ablation procedures, including photorefractive keratectomy (PRK) and laser epithelial keratomileusis (LASEK). Methods: In this interventional, comparative, follow-up study, 342 eyes of 171 patients were followed up for 6 months. Patients undergoing PRK or LASEK were included and were divided into two groups: group one (188 eyes of 94 patients) with an ablation depth of ? 65 µm and who received intraoperative 0.02% MMC for 30 s, and group two (154 eyes of 77 patients) with an ablation depth of < 65 µm and who received balanced salt solution for 30 s. Changes in endothelial cell density (ECD), central corneal thickness (CCT), coefficient of variation (CV), and hexagonality values were compared between the groups at 3 and 6 months after surgery. Results: The mean ± standard deviaiton (SD) age of the patients was 28.11 ± 6.56 years. The mean ± SD ECD did not change significantly in either group between the baseline and at 3 and 6 months postoperatively. The baseline mean ECD was significantly higher in group one than that in group two (P < 0.001) and remained so at 3 (P = 0.002) and 6 months (P = 0.022) postoperatively. The baseline hexagonality value was lower in group one (P = 0.173), with a gradual decrease during the postoperative follow-up as compared with that in group two (P = 0.016 and 0.001 at 3 and 6 months postoperatively, respectively). Group one had a significantly lower CCT at 3 and 6 months postoperatively (both P < 0.001) and a higher mean CV (3 months: P = 0.028; 6 months: P = 0.328). Conclusions: A single intraoperative application of MMC for 30 s as prophylaxis for corneal haze development during refractive surface ablation procedures had no significant effect on ECD up to 6 months postoperatively. Future studies with a contralateral-eye design (to neutralize factors specific to the individual patient), a larger sample size, and longer follow-up are necessary to confirm or disprove our observations.