AIM: To study the effect of mitomycin C (MMC) applied during transepithelial photorefractive keratectomy (TPRK) on the corneal endothelium one week (W1) and three months (M3) after surgery and its determinants. METHODS: In this two-armed cohort study conducted in 2022, eyes treated with MMC during TPRK (group 1) were compared with eyes not treated with MMC (group 2). The corneal endothelial cell (EC) count, EC density (ECD; cells/mm2), average (μm2), standard deviation (μm2), coefficient of variation (CV%), ECmax, ECmin, and EC percentage of hexagonality were estimated at W1 and M3. The postoperative changes in the EC count in the two groups were compared and correlated with the other independent variables. RESULTS: Group 1 had 26 eyes, and group 2 had 78 eyes. All TPRK indices were significantly higher for the eyes in group 1 than for those in group 2. The MMC usage was not a significant predictor of the change in ECD (P=0.644), change in CV (P=0.374), and change in the percentage of hexagonality of EC (P=0.164) at W1. However, the use of MMC was a significant predictor of changes in CV (P=0.014) and the change in the percentage of hexagonality of EC (P=0.039) at M3. The duration of laser exposure and the size of the optical zone influenced the correlation of MMC use with the changes in EC indices, postoperatively. CONCLUSION: The use of MMC doesn’t affect ECD, CV, and percentage of hexagonality at W1 if other surgical indices are considered. At M3 after operating myopic eyes by TPRK, MMC significantly influence the CV and percentage of hexagonality. The duration of the laser application and the size of the optical zone should be considered when determining the effect of MMC on the EC indices.