Knowledge about temporal associations for screen-detected gallstone disease and cardiovascular disease is limited. The objective of this study was to determine if screen-detected gallstones or cholecystectomy was associated with development of cardiovascular disease. A cohort study of three randomly selected groups from the general population of Copenhagen was performed. Participants (n = 5928) were examined 1982-1992 and underwent abdominal ultrasound examination to detect gallstone disease and were not informed of their gallstone status. Participants were followed up for occurrence of cardiovascular disease through nationwide registers until December 2014. Multivariable Cox regression analyses were performed including traditional cardiovascular disease risk factors and apolipoprotein E genotype. Gallstone disease was identified in 10% (591/5928) of participants at baseline of whom 6.8% had gallstones and 3.2% had cholecystectomy. The study population was followed for a period of 32 years with only 1% lost to follow-up. Gallstone disease was associated with all cardiovascular disease (hazard ratio (HR) 1.36, 95% confidence interval (CI) [1.17;1.59]) and to the subgroups coronary artery (HR 1.34, 95% CI [1.10;1.64]), cerebrovascular (HR 1.22, 95% CI [0.97;1.52]), and peripheral artery disease (HR 1.57, 95% CI [1.15;2.13]). No differences in estimates were identified for gallstones detected at ultrasound or cholecystectomy. Adjustment did not change estimates substantially. Gallstone disease seems to be independently associated to cardiovascular disease. Associations cannot be explained through traditional cardiovascular disease risk factors, apolipoprotein E4 allele, or detection bias. Future studies should explore the link between gallstone and cardiovascular disease further and gut microbiota may be a candidate mechanism.