An inconsistent association has been found between gallbladder disease and diabetes mellitus. We hypothesized that insulin resistance rather than diabetes status may be a primary factor involved in gallstone formation. A total of 5,653 adult participants in the third United States National Health and Nutrition Examination Survey without known diabetes underwent gallbladder ultrasonography and phlebotomy after an overnight fast for measurement of serum insulin, C-peptide, and glucose. Gallbladder disease was defined as ultrasound-documented gallstones or evidence of cholecystectomy. Subjects were characterized as having normal fasting glucose (<110 mg/dL), impaired fasting glucose (110 to <126 mg/dL), or undiagnosed diabetes (>/=126 mg/dL). After controlling for other known gallbladder disease risk factors, among women, undiagnosed diabetes was associated with increased risk of gallbladder disease (prevalence ratio [PR] = 1.91, 95% confidence interval [CI] = 1.29-2. 83); whereas impaired fasting glucose was unassociated. Gallbladder disease risk in women increased with levels of fasting insulin (PR = 1.63, 95% CI = 1.11-2.40) and C-peptide (PR = 2.07, 95% CI = 1.32-3. 25) comparing highest to lowest quintiles. However, the association of gallbladder disease with undiagnosed diabetes was not diminished when the model included fasting insulin (PR = 1.85, 95% CI = 1.24-2. 77). In men, there was a statistically nonsignificant association with undiagnosed diabetes (PR = 2.11, 95% CI = 0.76-5.85), but no association of gallbladder disease with insulin or C-peptide. Among women higher fasting serum insulin levels increased the risk of gallbladder disease, but did not account for the increased risk in persons with diabetes.