Gallstone disease in middle-aged women has been increasing due to changing dietary and environmental factors varying from country to country. The aim of the study was to determine the risk factors associated with gallstone disease in women aged ≥35 years of the district of Peshawar, Pakistan.A hospital-based case–control study.One hundred and ten women (55 cases and 55 controls) attending the Surgical Departments of two Government hospitals were enrolled for the study. All subjects were screened for ultra-sonography; biochemical and anthropometric measurements. They were interviewed for their past medical history; physical activity; 24-hr dietary recall and for demographic and socio-economic characteristics. Data were analyzed using Student's t-test, chi-square and multivariate conditional logistic regression to determine mean differences between the continuous variables; establish association between the categorical variables and to determine risk factors associated with gallstone disease, respectively.Of 55 cases, 15 (27%) had a family history of gallstone disease. Thirty five percent of the cases had a single calculus while 65% had multiple calculi with mean size of 14.85 ± 14.46 mm. Conditional logistic regression analysis demonstrated that the body mass index was the most significant risk factor for women's gallstone disease. The adjusted odds ratio for women's BMI ≥ 25 kg/m 2 was 2.93 (95% CI: 1.43–6.01), indicating almost a three times higher risk of gallstone disease than women with BMI < 25 kg/m 2 . The risk of gallstone disease was higher for women with low vitamin C intake (OR = 0.27; 95% CI: 0.08–0.91) and low physical activity (OR = 0.48; 95% CI: 0.24–0.96) than women with more physically active (score > 1.3) and having dietary vitamin C intake ≥ 75 mg per day.High body mass index, physical inactivity and low vitamin C intake are associated with gallstone disease in Pakistani women. Longitudinal studies are needed to confirm these findings.