OBJECTIVES: Aromatase inhibitors are the first-choice drugs for the treatment of hormone sensitive breast cancer. However, in addition to the scarcity of studies, there are controversies about their effects on vaginal epithelial cell proliferation in rats, especially those in persistent estrus. METHODS: To investigate vaginal epithelial cell proliferation by Ki-67 antigen expression, persistent estrus was induced in 42 randomly selected rats. These rats were randomly divided into 2 groups: group I (control, n=21), which received 0.1 mL of propylene glycol (vehicle) daily, and group II (experimental, n=21), which received 0.5 mg/kg or 0.125 mg/day of anastrozole diluted with 0.1 mL of propylene glycol. RESULTS: Light microscopy showed a higher concentration of cells with brown Ki-67 stained nuclei in the control compared to the experimental group. The mean percentage of Ki-67 stained nuclei per 500 cells in the vaginal epithelium was 68.64±2.64 and 30.46±2.00 [mean±standard error of the mean (SEM)] in the control and experimental groups, respectively (p<0.003). CONCLUSION: This study showed that anastrozole, at the dose and treatment duration selected, significantly decreased cell proliferation in the vaginal mucosa of the rats in persistent estrus.