To accurately measure the vaginal mucosa thickness across different age groups using histopathologic techniques and investigate the factors that may influence the thickness changes. This study aims to provide clinicians with objective evidence of variations in vaginal mucosal thickness, facilitating personalized medical decisions for patients. A retrospective analysis was conducted on clinical data from 348 patients who underwent local vaginal wall resection at the West China Second University Hospital, Sichuan University, from January 2021 and May 2022. The thickness of vaginal mucosa, epithelium and lamina propria was measured precisely under the microscope. And the 10th, 25th, 50th, 75th, and 90th percentile values of vaginal mucosa thickness across different age groups were counted and charted a dot-line plot. The percentile values for vaginal mucosa thickness exhibited a decreasing trend with increasing age; vaginal mucosa thickness showed significant correlations with times of delivery (P = 0.031) and age (P < 0.001), both of which were negatively associated. And vaginal mucosa thickness demonstrated no significant correlation with body mass index (BMI) (P = 0.325), times of abortions (P = 0.511), times of gestation (P = 0.101), menstrual cycle (P = 0.533), or types of delivery (P = 0.056); epithelial thickness showed significant associations with age (P < 0.001) and types of delivery (P = 0.017), both of which were negative correlations. Moreover, BMI (P = 0.429), times of abortions (P = 0.764), delivery (P = 0.079), gestation (P = 0.475), and menstrual cycle (P = 0.950) were nonassociated with epithelial thickness; lamina propria thickness displayed a significant correlation only with age (P = 0.002), and there were no obvious correlations observed between lamina propria thickness and BMI (P = 0.374), times of abortion (P = 0.417), delivery (P = 0.053), gestation (P = 0.101), types of delivery (P = 0.132) and menstrual cycle (P = 0.495). Moreover, when the age segmentation was thresholded at 35 and 50 years, both epithelial thickness and vaginal mucosa thickness were significantly correlated with age (P < 0.05). Lamina propria thickness was associated with age when the age threshold was set at 35 years (P = 0.007), whereas it showed no strong link with age when the age threshold was 50 years (P = 0.072). This study has innovatively established percentile reference values for vaginal mucosa thickness based on histopathology, furnishing clinicians with objective evidence of variations in vaginal mucosal thickness to facilitate personalized medical decisions for patients. The findings demonstrated a strong link between vaginal mucosa thickness and age, with epithelium likely playing a predominant role, while the association with lamina propria appeared to be less significant. Further research involving a larger sample size is warranted to elucidate the potential relationship with the lamina propria.