Gallstone disease is highly prevalent in the general population and is a major gastrointestinal cause of hospital admissions. The objectives were to determine whether circulating levels of 25-hydroxyvitamin D were associated to ultrasound proven gallstones or cholecystectomy in a general population sample. Determinants of vitamin D status were also explored. A random sample of 4130 people from the population of Copenhagen with ages 41-71 years were invited (N?=?4130) and 2650 participants were included. Ultrasound examinations were performed to assess gallstone status and blood samples were drawn to assess 25-hydroxyvitamin D and biomarkers of renal and hepatic function. Gallstone disease was found in 422 participants. Associations were estimated by logistic regression models. Levels of 25-hydroxyvitamin D was not significantly associated with gallstone disease. Time of birth during low vitamin D exposure was associated with gallstone disease (gallstone prevalence 18.0 versus 14.4?%, odds ratio 1.33, 95?% confidence interval [1.07; 1.65]). Highest quartile of cystatin C was significantly associated with gallstone disease (gallstone prevalence 22.1 versus 12.0?%, odds ratio 1.53, 95?% confidence interval [1.08; 2.18]). Serum levels of creatinine and alanine amino transferase were not associated with gallstone disease. Sensitivity analyses excluding participants with cholecystectomy did not alter results significantly. No association between 25-hydroxyvitamin D and gallstone disease was identified. Findings suggest gallstones to be associated to low vitamin D exposure in utero and to renal failure suggesting that vitamin D might have an impact on gallstone disease. Future studies should explore associations for vitamin D and gallstone disease prospectively.