Background Despite the fact that the majority of gallstones formed in the gallbladder are mainly composed of cholesterol, as they are formed from cholesterol-supersaturated bile, there is still no consensus regarding the contribution of blood serum lipids in the pathogenesis of gallbladder stone disease (GSD). Here, we aimed to investigate the relationship between hypercholesterolemia and the risk of new-onset asymptomatic GSD, and to determine the prevalence of factors associated with new-onset asymptomatic GSD in patients with hypercholesterolemia.Methods In this case-control study, we enrolled 927 Chinese patients with new-onset asymptomatic gallstone disease and 845 healthy controls starting in August 2012. Patients were matched for age, gender, race, occupation, systolic blood pressure, diastolic blood pressure, and fasting blood glucose levels (FBG). Body mass index, nonalcoholic fatty liver disease (NAFLD) and serum lipids indexes were compared and the relationships between BMI, blood lipid and gallbladder stone hazards were examined by using logistic multivariate regression models.Results The result showed a significant higher morbidity with GSD in hypercholesterolemia than non-hypercholesterolemia patients (X2=17.211, P<0.001). Of hypercholesterolemia patients, LDL-c (OR=1.493, P=0.029) and NAFLD (OR=2.723, P=0.022) were significant risk factors for GSD, while male sex (OR=0.244, P=0.033), weight (OR=0.961, P=0.022), HDL-c (OR=0.305, P<0.001), and FBG (OR=0.687, P=0.034) significantly negatively correlated with GSD in univariate analysis. Multivariate logistic regression indicated weakly positive correlations with NAFLD (OR=3.284, P=0.054), and significant negative correlations with weight (OR=0.930, P=0.018), HDL-c (OR=0.144, P<0.001), and GSD. Conclusion Hypercholesterolemia acts as an independent risk factor for new-onset asymptomatic GSD, while obesity and NAFLD are synergistic factors. Interestingly, we are the first to report that elevated weight was inversely associated with GSD in patients with hypercholesterolemia.