This study aimed to evaluate the association between serum vitamin C and pelvic organ prolapse (POP) in middle-aged and elderly women. The 2005-2006 National Health and Nutrition Examination Survey (NHANES) database was used for cross-sectional study. The quartile method was employed to stratify the concentration of vitamin C. A serum vitamin C and POP weighted logistic regression model was constructed by adjusting for different confounding factors. All confounding factors were adjusted for interaction tests. Subgroup analysis was conducted to delve into the correlation of confounding factors with significant interaction term P-values. A total of 760 samples were included in this study. There was a negative correlation between serum vitamin C and POP (OR=0.48, 95%CI: 0.23-0.99, P<0.05), showing that as the concentration of vitamin C increased, the risk of POP tended to decrease. Race, poverty-to-income ratio (PIR), alcohol consumption, hysterectomy, and diabetes can greatly influence the association between the two (P for interaction <0.05). Especially in populations with a PIR of 1.3-3.5 (OR=0.05, 95%CI: 0.01-0.28, P<0.001), no alcohol consumption (OR=0.17, 95%CI: 0.07-0.46, P<0.001), and no hysterectomy (OR=0.27, 95%CI: 0.08-0.85, P<0.05), there was a strongly negative correlation between serum vitamin C and the risk of POP. In middle-aged and elderly women, there is a strong negative correlation between serum vitamin C and the risk of POP, especially in populations with a PIR of 1.3-3.5, no alcohol consumption, and no hysterectomy.