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High levels of cholesterol have been associated with certain gallbladder disorders such as cholesterolosis and gallstone disease. Furthermore, obesity is considered the main risk factor for cholesterol gallstone disease. We investigated the incidence of cholesterolosis in patients with and patients without gallbladder stones (GS). We reviewed the clinical records of patients with gallstone disease and other gallbladder disorders who had consecutive cholecystectomy during a 5-year period. We recorded demographic data, sex, age, serum cholesterol levels, and body mass index. The diagnosis of cholesterolosis was made macroscopically and microscopically. A total of 636 patients were included in this study: 446 with and 190 without GS. Cholesterolosis was more frequent in patients without GS (p < 0.01). However, hypercholesterolemia occurred more frequently in patients with GS (p < 0.001). Obese patients with GS had higher percentages of cholesterolosis and hypercholesterolemia than did eutrophic patients (p < 0.01 and p < 0.05, respectively). We suggest that cholesterolosis in the human gallbladder is not necessarily associated with gallstone disease and high plasma cholesterol levels.