Studies showed that vitamin D (25-hydroxyvitamin D) level in the human blood circulation could be affected by exogenous estrogen exposure. This study aims to explore the relationships between urinary phytoestrogens metabolites and serum total 25(OH)D in general population, urinary phytoestrogens metabolites (daidzein, enterodiol, enterolactone, equol, genistein and o-desmethylangolensin). Totally 2,609 adults 6 y old from the 2009-2010 National Health and Nutrition Examination Surveys (NHANES) were recruited into the cross-sectional analyses and information including demographic, socioeconomic, examinations and laboratory test were collected. All analyses were performed using Stata13.0, one-way analysis of variance and multivariable regression were utilised according to data characteristics, respectively. It showed that age, race, education level, body mass index (BMI), and sampling season had significant effects on serum 25(OH)D level (all p,0.001). In the whole population, urinary enterodiol and equol were significantly positively associated with serum total 25(OH)D level (b50.86, 95%CI50.08-1.65, p,0.05; b51.68, 95%CI50.91-2.45, p,0.001). Equol was also found significantly positively correlated with total 25(OH)D in both female and male separately (b51.69, 95%CI50.51-2.87, p,0.05; b51.66, 95%CI50.63-2.69, p,0.05). Phytoestrogen concentrations in the urinary and 25(OH)D levels in the serum had proved a positive correlation in our study, which provide theoretical basis and reference for the dietary nutrient intake in the population.