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Purpose: To evaluate whether prostaglandin (PG) analogue use is associated with alterations in keratocyte density and central corneal thickness (CCT) in subjects with primary open-angle glaucoma (POAG). Materials and Methods: Thirty-five POAG patients treated with PG analogues for >2 years and 35 control subjects without glaucoma were included in this cross-sectional study. All subjects were underwent CCT measurements using ultrasound pachymetry. Keratocyte densities of each stromal layer were determined by in vivo confocal microscopy. Student′s t-test and Chi-square test were used for statistical evaluations. Correlations between keratocyte densities and CCT were analyzed using Pearson′s correlation analysis. Results: Keratocyte densities in each stromal layer were significantly lower in glaucoma patients receiving PG analogues as compared to those of controls (P < 0.001). The mean CCT was also lower in glaucoma patients (515.2 ± 18.8 μ) than control subjects (549.6 ± 21.1 μ, P < 0.001). A positive correlation between keratocyte densities in each stromal layer and CCT was observed in POAG patients. Conclusions: Long-term administration of topical PG analogues may adversely influence keratocyte densities and CCT. Further prospective studies are required clarify the relationship between PG analogues and their effects on the cornea.