BACKGROUND: The relationship between serum vitamin D status and urinary leakage (UL) among middle-aged females needs to be further studied. The aim of this study was to evaluate the association of serum 25-hydroxyvitamin D [25(OH)D] concentrations with UL among American females ages 45 years and over. METHODS: Seven cycles of the National Health and Nutrition Examination Survey (NHANES) with self-report UL data, were used. A total of 9525 women aged 45 years and older were enrolled in this study. Univariate and multivariate logistic regression models and the smooth curve fitting were utilized to analyze the association between clinical UL and serum 25-hydroxyvitamin D [25(OH)D] concentrations. RESULTS: A non-linear relationship between serum 25(OH)D concentrations and clinical ULwas observed. When serum 25(OH)D concentration was higher than the inflection point 63.5 nmol/L, a positive correlation was observed between serum 25(OH)D concentrations and clinical UL ([OR]: 1.007, 95%CI: 1.005-1.009, P < 0.01). However, when serum 25(OH)D concentration was below the inflection point 63.5 nmol/L, a negative correlation was observed between serum 25(OH)D concentrations and clinical UL ([OR]: 0.993, 95%CI: 0.989-0.996, P < 0.01). CONCLUSIONS: The association between serum vitamin D and the risk of UL exhibited a U-shaped pattern among US middle-aged females, with an inflection point occurring at a serum 25(OH)D concentration of 63.5 nmol/L.